CN100572555C - Use the amplicon melting analysis of saturable dye - Google Patents

Abstract

Methods for nucleic acid analysis are provided wherein an at least partially double-stranded target nucleic acid is mixed with a double-stranded DNA binding dye having a saturation percentage of at least 50% to form a mixture. In one embodiment, said nucleic acid is amplified in the presence of said double-stranded DNA-binding dye, and in another embodiment, as said mixture is heated, by measuring The fluorescence generates a melting curve against the target nucleic acid. Dyes for use in nucleic acid analysis and methods of making the dyes are also provided.

Description

Use the amplicon melting analysis of saturable dye

Invention field

The present invention relates under the condition that the double-strandednucleic acid combination dye exists, carry out the method for foranalysis of nucleic acids.

Background of invention

The method of analyzing DNA sequence variations can be divided into two classes substantially: the scanning that the unknown the is made a variation 1) gene type and 2 that known array is made a variation).The method of existing multiple known array mutant gene somatotype, and can adopt the one step that uses fluorescent probe, method homogeneity, stopped pipe (Lay MJ etc., Clin.Chem 1997; 43:2262-7).On the contrary, most of scanning technique at the unknown variation requires PCR gel electrophoresis or post afterwards to separate.These technology comprise single strand conformation polymorphism (Orita O etc., Proc Natl Acad Sci USA1989; 86:2766-70), heteroduplex molecule migration (Nataraj AJ etc., Electrophoresis 1999; 20:1177-85), denaturing gradient gel electrophoresis (Abrams ES etc., Genomics 1990; 7:463-75), temperature gradient gel elec-trophoresis (TGGE) (Wartell RM etc., J Chromatogr A 1998; 806:169-85), enzyme or chemical chop method (Taylor GR etc., GenetAnal 1999; 14:181-6) and dna sequencing.Also require PCR a plurality of steps afterwards by the new sudden change of order-checking identification, order-checking and gel electrophoresis circulation just.Sex change high performance liquid chromatography (Xiao W etc., Hum Mutat 2001; 17:439-74) relate to PCR product injection chromatography column.

Recently, the existing report of homogeneity fluorescent method that is used to suddenly change and scans.

Green I (MolecularProbes, Eugene, Oregon) be a kind ofly in PCR in real time, form through being usually used in monitoring product (WittwerCT etc., BioTechniques 1997; 22:130-8) and melting temperature(Tm) (Ririe KM etc., Anal.Biochem1997; Double-stranded specific DNA dyestuff 245:154-60).By using

The curve analysis of GreenI has detected (LipskyRH etc., the Clin Chem 2001 of existing of the single sequence change of heterozygote in interior product at 167bp; 47:635-44).Yet amplification back had been carried out purifying and had been added high density the PCR product before melting analysis

Green I.In the described method in order to detect

Green I concentration suppresses PCR reacts (Wittwer CT etc., BioTechniques 1997; 22:130-1,134-8); Thereby this dyestuff adds after amplification.Hope can have a kind of can be used to the detect existence of the single sequence change of heterozygote and the dyestuff that can add before PCR.

Single nucleotide polymorphism (SNPs) is observed modal heritable variation in the middle of human and other species so far.In these polymorphisms, the change of single base is only arranged between the individuality.Described change may cause in the albumen an amino acid whose change, change transcription speed, influence the mRNA montage or the pair cell process does not obviously act on.Sometimes under the situation of described change reticent (for example, not having in its amino acids coding under the situation of change), the SNP gene type may still have value, if described change and the distinct performance type that is caused by another hereditary change link to each other (related).

The method of existing multiple SNP gene type.Most of PCR or other amplification techniques of adopting is with the interested template that increases.Can use the synchronous or subsequent analysis technology that comprises gel electrophoresis, mass spectrum and fluorescence.Because it is homogeneity, and need not add reagent in amplification beginning back or for analysis to the manual sampling of reactant, fluorescence technique is attractive.Exemplary similar technology uses Oligonucleolide primers location interesting areas and fluorescent mark or dyestuff to be used to produce signal.By using the stable thermophilic enzyme of DNA denaturation temperature, the method for exemplary PCR-based is complete stopped pipe, after the heating beginning, does not need additional step like this.Can adopt fluorescence PCR method several stopped pipes, homogeneity for the SNPs gene type.These methods comprise uses FRET oligonucleotide probe (the contiguous hybridization probe that two interactional luminophores are arranged, the TaqMan probe, Molecular Beacons, single oligonucleotide probe (the G-cancellation probe that Scorpions), a fluorophor is only arranged, Crockett, A.O. and C.T.Wittwer, Anal.Biochem.2001; 290:89-97 and SimpleProbes, Idaho Technology) and the technology of using double-stranded DNA dyestuff rather than the fluorescently-labeled oligonucleotide probe of covalency.Owing to do not need the oligonucleotide probe of mark, can reduce design time and detect cost, described dyestuff technology is attractive.

Two kinds of SNP typing methods that use the double-stranded DNA dyestuff are disclosed.Exist the allele specific amplification under the double-stranded DNA dyestuff situation can be used to PCR in real time gene type (Germer S etc., Genome Research 2000; 10:258-266).In the method for the reference of Germer, under the situation that has a total reverse primer, two amplification of allele-specific primers difference one or another allelotrope that 3 '-base there are differences.Though do not need fluorescently-labeled oligonucleotide, gene type requires 3 primers and at every kind of genotypic two reaction tanks.In addition, the PCR in real time instrument that needs each circulation fluorescent signal of monitoring.

Another kind of method based on dyestuff does not need real-time monitoring, and every kind of SNP genotype only needs a reaction tank, and uses melting analysis (Germer S etc., Genome Research 1999; 9:72-79).In this method, as the Germer method of front, also used the allele specific amplification that needs 3 primers.In addition, one in the described primer contains GC-hair clip afterbody, and to improve the melting temperature(Tm) of an amplicon, making in a reaction tank to distinguish by melting temperature(Tm) becomes possibility.Detect fluorescence behind the pcr amplification, and do not need real-time acquisition.

Summary of the invention

One aspect of the present invention provides a kind of method that only needs conventional PCR reagent, primer and simply add " saturated " double-stranded DNA combination dye before PCR.For the purposes of the present invention, " saturated " dyestuff is meant that this dyestuff thinks when not existing under the dyestuff situation double-stranded DNA amount (for example about 10 nanograms/microlitre) that is produced usually by PCR to provide the concentration of maximum fluorescence signal to exist, and can obviously not suppress the PCR reaction.Though described dyestuff during by its saturation concentration and the compatibility of PCR differentiate, it will be appreciated that described dyestuff can use with much lower concentration.In the amplification procedure or follow closely after the amplification, described dyestuff can with applying marking primer situation under similarly mode be used to distinguish heteroduplex molecule and homoduplex molecule by curve analysis.The discriminating of heteroduplex molecule and homoduplex molecule can be used to comprise the multiple analysis of sudden change scanning and SNP gene type.Term " scanning " is meant a nucleic acid fragment and one with reference to the nucleic acid fragment comparison to detect the process that exists of any difference on the sequence.Expression exists the positive findings of sequence difference not necessarily to reflect definite character or its position on nucleic acid fragment of sequence variations.Term " gene type " comprises detection and determines known variant nucleic acid sequence that these variations include but not limited to that SNPs, base deletion, base insertion, sequence repetition, rearrangement, inversion, base methylate, short series connection repeat number; And under the genomic situation of twice body, whether genome is the homozygote or the heterozygote of sequence variations, and the cis/trans position relation (haplotyping) of two or more sequence variations on DNA chain.

Another aspect of the present invention has been differentiated multiple double-stranded DNA combination dye.Double-stranded DNA combination dye of the present invention can exist with the abundant saturation capacity for DNA in amplification or after the amplification, and the inhibition to PCR simultaneously minimizes.For example, under the compatible concentration of maximum PCR, the double-stranded DNA combination dye has and is at least 50% saturated per-cent.In other embodiments, described saturated per-cent is at least 80%, more specifically is at least 90%.In also having other embodiments, described saturated per-cent at least 99%.It will be appreciated that described saturated per-cent is the fluorescence per-cent of comparing with the fluorescence of the identical dyestuff of saturation concentration (concentration of possible high fluorescent just is provided) under the situation that has predetermined double-stranded DNA amount.For instance, predetermined double-stranded DNA amount is 100 nanograms/10 microlitres, and this is the DNA amount that typical PCR produces when arriving plateau in latter stage.Need understand further that dye formulations may comprise the impurity of suppression of amplification.Such impurity should be removed before definite saturated per-cent.Also it will be appreciated that, the measurement of saturated per-cent fluorescence intensity is being carried out with the wavelength place of detecting the dyestuff matched well that combines double-stranded DNA, and if possible, do not detect at the wavelength place that can detect from the high background fluorescence of the free dye or the dyestuff bonded secondary form (for example, dyestuff combines with double-stranded DNA-dye composition or combines with single-chain nucleic acid) that under high dyestuff-base pair ratio, may form.

Also have aspect another of the present invention, the double-stranded DNA combination dye has the saturation ratio greater than 50% under the compatible concentration of maximum PCR, and has possibility and the incompatible excitation/emission spectrum of conventional PCR in real time instrument.Being used for " routine " instrument that PCR in real time analyzes has between the excitation wavelength of about 450-490 nanometer and long between the emitting detection wave of about 510-530 nanometer.Have been found that some " blueness " dyestuff and these system compatibles, though its excitation/emission spectrum may be different.Like this, in this aspect of the invention, provide in a kind of PCR process or followed the method for using after the PCR conventional PCR in real time instrument and double-stranded DNA combination dye to analyze closely, wherein the double-stranded DNA combination dye is as the nanometer between 410-465 that has that records in having the PCR damping fluid of double-stranded DNA, especially between the maximum excitation wavelength of 430-460 nanometer, and have a nanometer between 450-500, especially between the maximum emission wavelength of 455-485 nanometer.Suitable instrument can use between above-mentioned exciting/detection zone, or can improve according to the excitation/emission peak value of dyestuff.Detect " blueness " dyestuff of the present invention and detect as fluorescein with

The suitable interval of conventional dyestuffs such as Green I can comprise the detection wavelength of the excitation wavelength and the 500-560 nanometer of 440-470 nanometer.

In one embodiment, described dyestuff is that a kind of discriminating is the dyestuff of LightCycler Green (also can be described as LCGreen).The synthetic of LC Green is described below, and the excitation/emission spectrum of LC Green has been described in Figure 11.The bells and whistles that has shown LC Green in the table 1.Similarly, differentiate to be that effectively other dyestuffs can use within the scope of the invention in the table 1.Though the definite structure of some dyestuffs is still the unknown in these dyestuffs, they are considered to asymmetric cyanine, and the multifrequency nature of these fluorescent core acid dyes has been described in table 1.

Though example provided here is at curve analysis, it will be appreciated that dyestuff of the present invention can be used for the multiple real-time quantitative PCR analysis that comprises the method for nucleic acid quantification, initial concentration mensuration, the detection to there being a kind of nucleic acid, the stack detection of using label probe and other PCR-based.

In addition, though that mention is PCR, other amplification methods can be compatible with dyestuff of the present invention.Suitable method like this comprises strand displacement amplification (SDA), depends on the amplification (NASBA) of nucleotide sequence, the amplification of cascade rolling circle amplification (CRCA), the mediation of Q β replicative enzyme, etc. the nucleic acid amplification (ICAN) that starts of gentle chimeric primers, the amplification (TMA) of transcriptive intermediate etc.Therefore, marquis when using term PCR need be interpreted as to comprise other alternative amplification methods.

In addition, need understand the double-stranded DNA combination dye and comprise intercalator and other and nucleic acid bonded dyestuff,, or otherwise produce difference signal based on double-strandednucleic acid quantity as long as described dyestuff difference is in conjunction with two strands and single-chain nucleic acid.

Like this, the present invention includes one or more double chain combined with dye that quantitative or qualitative amplification is analyzed that are used for described here.In one aspect of the invention, provide a kind of PCR reaction mixture, Oligonucleolide primers that this mixture comprises target nucleic acid, PCR reagent, be provided with for amplifying target nucleic acid and double-stranded DNA combination dye with saturated per-cent of at least 50%.

In another aspect of the present invention, provide the method for foranalysis of nucleic acids.In one embodiment, a kind of method of gene type is provided, this method is included in amplifying target nucleic acid under the double-stranded DNA combination dye existence condition with at least 50% saturated per-cent, and the target nucleic acid of fusion amplification to be generating melting curve, and by steps such as melting curve sldh gene types.In another embodiment, a kind of sudden change method for scanning is provided, this method comprises that (a) adds the double-stranded DNA combination dye with at least 50% saturated per-cent to the sample that contains target nucleic acid, (b) amplifying target nucleic acid under the condition that described double-stranded DNA combination dye exists, (c) target nucleic acid of fusion amplification is to generate melting curve, repeating step (b) and on second sample, and step such as more described melting curve (c) obtaining the second melting curve.In also having another kind of embodiment, a kind of method of pcr analysis is provided, this method comprises the sample mix of the double-stranded DNA combination dye that will have at least 50% saturated per-cent and the primer that comprises target nucleic acid and be provided with for amplifying target nucleic acid, amplifying target nucleic acid under the condition that described double-stranded DNA combination dye exists, and the steps such as fluorescence of monitoring described double-stranded DNA combination dye.Monitoring can occur in the amplification procedure, the amplification after or both.

Aspect another one in addition of the present invention, the step that comprises a kind of pcr analysis method is provided, comprise the sample mix of double-stranded DNA combination dye with the primer that contains target nucleic acid and be provided with for amplifying target nucleic acid, amplifying target nucleic acid under the condition that described double-stranded DNA combination dye exists, monitor the fluorescence of described double-stranded DNA combination dye, generation is at the melting curve of target nucleic acid, the stdn melting curve, repeat mixing, amplification, curve with another target nucleic acid at least and produce and standardised step, and the steps such as melting curve of standard of comparisonization.

In an additional aspect of the present invention, a kind of method of foranalysis of nucleic acids is provided, this method comprises that the target nucleic acid of near small part two strands mixes with the double-stranded DNA combination dye with at least 50% saturated per-cent to form mixture, and, produce the steps such as melting curve of described target nucleic acid by the fluorescence of measuring described double-stranded DNA combination dye along with mixture is heated.

Aspect further one of the present invention, provide the test kit of Oligonucleolide primers that comprises amplifing reagent, is provided with for amplifying target nucleic acid and double-stranded DNA combination dye with at least 50% saturated per-cent.Can use any dyestuff discussed herein in the described test kit.

As described herein-in, can use multiple double-stranded DNA combination dye in the embodiments of the present invention.

According to the detailed description of following illustrated embodiment, supplementary features of the present invention will be conspicuous for being proficient in those skilled in the art.

Description of drawings

Fig. 1 has shown the gene type that uses the factor V Leiden of LightCycler Green.The negative first order derivative that has shown melting curve (dF/dT).

Fig. 2 has shown the influence that speed of cooling detects heteroduplex molecule before the melting analysis.

Fig. 3 has shown the influence that rate of heating detects heteroduplex molecule in the melting analysis process.

Fig. 4 has shown the model system that detects 6 kinds of combinations of heteroduplex molecule.

Fig. 5 A-D has shown the comparison of methods of genotyping; Fig. 5 A has shown the cystic fibrosis synoptic diagram, wherein the position of optional markings marks with star (★) on primer, Fig. 5 B has shown the gene type of applying marking primer, and Fig. 5 C has shown the gene type that uses LightCycler Green, and Fig. 5 D has shown use

The gene type of Green I attempt (homozygote:----wt,---F508del, heterozygote:--F508del,---I507del,----F508C).

Fig. 6 shown on longer amplicon the gene type that uses LightCycler Green (----homozygote (TT),---homozygote (CC),--heterozygote (TC)).The melting curve that has shown three individualities (not being derivative).

Fig. 7 A-B has shown use

The derivative melting curve of the DNA mixture of Green I (Fig. 7 A) and LightCycler Green (Fig. 7 B).

Fig. 8 has shown that there is the change in fluorescence of non-linear hour in broad variety DNA sample.Shown on the figure LightCycler Green (zero) and

GreenI (■).

Fig. 9 A-B has shown the dyestuff titration measuring saturated per-cent, among Fig. 9 A: ◆-

Green, ■-

Gold, ▲-Pico Green; Among Fig. 9 B: zero-LightCycler Green, ■

Green.For

The exemplary PCR scope of Green and LightCycler Green is represented with dash box.

Figure 10 has shown the influence of dye strength to melting temperature(Tm).

Figure 11 A-B shown LightCycler Green (Figure 11 A) and Green I (Figure 11 B) excites and emmission spectrum.

Figure 12 A-D shown on the fragment of beta Globulin 110bp 6 kinds of different genotype (--SS,---AA,----CC,--SC ... AC,---AS) the high resolving power curve analysis of quadruplicate sample.Figure 12 A has shown the raw data of unwinding and obtaining from the high resolving power of every kind of quadruplicate sample of genotype; Figure 12 B has shown the high resolving power melting curve through standardization of 6 kinds of quadruplicate samples of genotype; Figure 12 C has shown that the temperature of 6 kinds of quadruplicate samples of genotype moves, standardized, high resolving power melting curve.Sample temperature moves with the fluorescence between overlapping 5% and 10%; Figure 12 D has shown the fluorescence difference curve that the data by Figure 12 C obtain.Every difference curve obtains to obtain variance data by every kind of sample of deduction from standard (AA) curve.When adopting quadruplicate sample, because the eclipsed relation is seeming to be less than 4 duplicate samples in some cases.

Figure 13 A shown the segmental 3 kinds of genotypic repeat samples of people's 5-hydroxytryptamine receptor 2A (HTR2A) gene 544bp curve analysis (---TC,---CC,----TT).Fluorescence part between the use 10% and 20% data has been carried out stdn and temperature moves processing.The theory of Figure 13 B. homoduplex molecule synoptic diagram that unwinds.Mononucleotide polymorphism site marks with (X).

Figure 14 has shown that cystic fibrosis strides the segmental 6 kinds of genotypic difference curves of film transduction regulatory (CFTR) gene 612bp.By the overlapping of fluorescence part between 30% and 40% figure has been carried out stdn and temperature moves processing, and from a kind of wild-type figure, deducted.

Figure 15 has shown the pedigree chart of the Utah family 1331 of CEPH reference.The HLA-A genotype of Utah family 1331 is as follows: A:02011; B:3101; C:2402101; D:03011; E:01011.Each individuality is numbered.Women's (circle); Male sex's (square frame).

Figure 16 A-B has shown 1331 members' of Utah family melting curve.In 17 family members, exist 6 kinds of genotypic 6 different melting curves representing the HLA-A exon 2.Figure 16 A has shown complete melting curve, and Figure 16 B has shown the part (being illustrated in the 16A square frame) of amplifying and indicated genotype and individuality in parenthesis.

Figure 17 shown by mixing determined two samples genotype (---BM15,----BM16,-----BM15+BM16).Two homozygote sample BM15 (0101) and BM16 (0201) have the difference of 15bp on the HLA-A exon 2.The melting curve of BM15 and BM16 is similar when detecting respectively, but when mixing, the mispairing of 15bp is offset melting curve.

Detailed Description Of The Invention

Because it shows the great variety of fluorescence in the PCR process,Green I be a kind of dyestuff that is widely used in melting analysis (Wittwer CT etc., Biotechniques 1997; 22:130-1,134-8; Wittwer CT etc. show " Real-Time PCR ", see the chief editors such as Persing D, Diagnostic Molecular Microbiology:Principles and Applications, ASM publishing house, 2004). It is contemplated that such dyestuff both can be used for homozygous Genotyping, can be used for again the scanning to the heterozygote sequence variation.

Green I at first in melting analysis in order to distinguish Tm value difference other 2 ℃ or more different PCR product (Ririe KM etc., Anal Biochem 1997; 245:154-160). Subsequently,Green I is used for differentiating base deletion (Aoshima T etc., Clin Chem 2000; 46:119-22), the genotype dinucleotides repeats (Marziliano N etc., Clin Chem 2000; 46:423-5) and differentiate that multiple sequence changes (Lipsky RH etc., Clin Chem 2001; 47:635-44; Pirulli D etc., Clin Chem 2000; 46:1842-4; Tanriverdi S etc., J Clin Microbiol.2002; 40:3237-44; Hladnik U etc., Clin Exp Med.2002; 2:105-8). Yet Tm difference may be very little and may challenge the resolution limit of existing instrument between the genotype. In fact, thinkGreenI " can not use " (von Ahsen N etc., Clin Chem 2001 in daily Genotyping is used; 47:1331-1332). The Tm value that adopts the melting curve Genotyping of normally used double-stranded specific DNA dyestuff to comprise to widen between the limited proportionality and improve along with unwinding (Douthart RJ etc., Biochemistry 1973; The compression (Fig. 5 D) of Tm difference 12:214-20) and between the genotype. These factors have reduced

Green I is used for distinguishing genotypic ability.

The amplification of heterozygote DNA produces 4 kinds of different strands, and these strands produce 2 kinds of homoduplex molecules and 2 kinds of heteroduplex molecules under sex change and cooling condition. In theory, all 4 kinds of products to have different Tm values and melting curve should be all 4 kinds of two strandss to strand change synthetic. Yet the double-stranded specific DNA dyestuff may redistribution in separating chain process (Aktipis S etc., Biochemistry 1975; 14:326-31), this causes dyestuff from low-melting heteroduplex molecule release and again is distributed on more dystectic homoduplex molecule. Because under the concentration compatible with PCR

That Green I does not have is saturated (Wittwer CT etc., Biotechniques 1997; 22:130-1,134-8; Fig. 9), such redistribution is disappearance consistent (Fig. 5 D) possible and that unwind and change with heteroduplex molecule.

LightCycler Green and other dyestuffs of the present invention can use in the application of Genotyping and scanning. Be in the homozygous situation in only increase a kind of PCR product and sequence, only form the homoduplex molecule. Use dyestuff of the present invention, the Tm value difference is different between the different homoduplex molecules does not have compressed (Fig. 5 C), and the obvious differentiation between the genotype is possible. The multi-products that reaction exists also can be used for be differentiated and be distinguished to dyestuff of the present invention, for example the homoduplex molecule of the amplification generation by homozygous a plurality of gene locus or a plurality of targets. On the contrary, the chances are because the redistribution of dyestuff is used

Green I in most of the cases can only observe minority product (seeing Fig. 7 A).

When the one or more heterozygote target of amplification, use dyestuff of the present invention can easily observe heteroduplex molecule. Detection also identifies that the ability of heteroduplex molecule is particularly useful for detecting heterozygote genotype and scanning unknown mutation. Adopt to be used for PCR in real time asGreen I、

Gold and ethidium bromide etc. can not be observed wherein, and the traditional double chain DNA dyestuff of heteroduplex molecule product is impossible realize this point.

In separating chain process, the homoduplex molecule can is combined and form to the chain of heteroduplex molecule again with its complete complementary chain. Because production concentration is very high when PCR finishes, this again cohesive process occur fast. Be near time of (especially being between heteroduplex and the homoduplex product Tm value) its melting temperature by the restriction product, described again combination can be minimized. Except separating the again combination of chain process medium chain, the selective cross of chain and its complementary strand that mate fully or mispairing is subject to the impact of cooling velocity. Here under the represented condition, cooling is conducive to heteroduplex molecule most and forms fast, and is being lower than under-0.1 ℃/second the speed, heteroduplex molecule often can disappear (Fig. 2). This is opposite with sex change HPLC technology, and the latter's cooling velocity much lower (0.01 to-0.02 ℃/second approximately) still effectively forms heteroduplex molecule (Xiao W etc., Hum Mutat 2001; 17:439-74). Perhaps, the relative velocity that homoduplex molecule and heteroduplex molecule form depends on the product size very much, and the result who uses little amplicon to obtain may not have typicalness for the larger product that more generally uses among the dHPLC.

By unwinding, spend more analysis time with lower speed, can improve the ability to see things in their true light between the homozygote genotype. DNA concentration is a source of potential error in the melting curve Genotyping on the impact of Tm. When using the amplicon of at random 100bp of 50%GC content under the PCR condition, the Tm value difference is different between the product of 0.05 μ M and 0.5 μ M is about 0.7 ℃ of (vonAhsen N etc., Clin Chem 2001; 47:1956-61; Wetmur JG, Grit Rev Biochem Mol Biol 1991; 26:227-59). When the genotypic Tm value of different homozygotes very near the time, this change may be important. Yet different PCR samples trend towards reaching plateau with same products concentration, and concentration difference is normally minimum after the amplification so. Simultaneously, can by real-time fluorescence estimate amplicon concentration and in addition higher Genotyping precision regulate the Tm value. In addition, can use asymmetric PCR with the final concentration of volitional check PCR product.

All single base heterozygotes and homozygote might be distinguished with LightCycler Green. In heterozygote detected, absolute melting temperature and DNA concentration as influencing factor did not resemble to use and relate to that to distinguish the genotypic method of homozygote so important. Heteroduplex molecule affects the shape of melting curve, " in early days " low temperature part that is especially changing. So that " late period " high-temperature part of the transformation of unwinding is overlapping, different melting curves can be Temperature Matching by the X-axis translation. Then can infer whether heteroduplex molecule exists with higher accuracy.

The accuracy of instrument no matter, some genotypic Tm value can be almost identical. A kind of method that detection has the homozygote variation of identical Tm value is that variant is mixed jointly. The heteroduplex molecule that obtains can unwind under the temperature lower than homozygote, shows in standardized melting curve in the decline of mainly unwinding before changing.

Thereby, using present utilizable pcr amplification device, LightCycler Green can differentiate in melting curve changes and can not use at presentThe heteroduplex molecule that Green I differentiates. Why illustrated among Fig. 7 A

Green I can not differentiate the possible cause that low temperature unwinds and changes easily. When the dna fragmentation that exists several stability to increase progressively, useThe low temperature peak of Green I is very little with comparing of use LightCycler Green. In separating chain process,

Green I may discharge from the low temperature duplex molecule, only links to each other with the two strands of unwinding under higher temperature. This causes each follow-up peak than previous height, even if so that the peak of minimum temperature can be observed also very little. As what see among Fig. 7 B, with LightCycler Green but be not by

Green I has easily detected the product that low temperature unwinds.

Use the advantage of LC Green to cause other and PCR is compatible and the discriminating that is suitable for the double-stranded DNA dyestuff of Genotyping under the compatible concentration of PCR. Many dyestuffs for method of the present invention belong to cyanine family. Cyanine dye is those dyestuffs that contain one or more divalence " C (R)=" part at two nitrogenous heterocyclic chains of connection. Group " R " may be hydrogen or any carbon substituting group, and for example hydrogen or alkyl comprises the C that can be optionally substituted_1-6Alkyl. It will be appreciated that each " R " can be independent the selection in cyanine dye of an above divalence is arranged therein " C (R)=" part. As by illustrative general formula described here further definition, such cyanine dye can be monomer or dimer. Except cyanine dye, estimate that other double-stranded DNA combination dye families also are useful in described PCR reactant mixture, the method and composition here, these families include but not limited to the phenanthridines intercalator and based on the metal intercalator of phenanthroline (phenanthroline-based).

Exemplary dyestuff useful in described PCR reactant mixture, method and composition comprises PO-PRO^TM-1、BO-PRO ^TM-1、

43、

44、 45、

Blue、 POPO ^TM-1、POPO ^TM-3、BOBO ^TM-1、BOBO ^TM-3、LO-PRO ^TM-1、JO-PRO ^TM-1、 YO-

-1、TO-

-1、 11、

13、

15、

16、

20、 23、TOTO ^TM-3、

-3(Molecular Probes，Inc.，Eugene， OR)、 (Cambrex Bio Science Rockland Inc., Rockland, ME), Thiazole Orange (Aldrich Chemical Co., Milwaukee, WI) and reactive monoazo dyestuffs G5, H5, D6, E6, P6, R6, Y6, Z6 and D8 described here.

The exemplary cyanine dye that here uses in described PCR reactant mixture, the method and composition also comprise have pyridine, monomer or dimer and those compounds of usually being described by structural formula I of the asymmetric cyanine of pyrimidine, quinoline, isoquinolin or purine core texture:

Structural formula I

Wherein

The part representative is chosen the condensed monocycle that replaces wantonly or is encircled aromatic nucleus or nitrogenous aromatic heterocycle more;

X is oxygen, sulphur, selenium, tellurium, or is selected from C (CH ₃) ₂And NR ¹Group, R wherein ¹Be hydrogen or alkyl, comprise C _1-6Alkyl and C _2-6Alkyl;

R ²Be alkyl, comprise C _1-6Alkyl and C _2-6Alkyl; Cycloalkyl comprises C _3-8Cycloalkyl; Aryl; Aralkyl comprises aryl (C _1-2Alkyl); Hydroxyalkyl; Alkoxyalkyl; Aminoalkyl; Alkyl monosubstituted amino alkyl and dialkyl aminoalkyl; The trialkyl ammonium alkyl; Alkyl glycosyl and aryl glycosyl; Alkane carboxylic acid amides and fragrant carboxylic acid amides; Alkyl sulphonyl and aryl sulfonyl; The thiazolinyl carboxylate radical; The thiazolinyl carboxylic acid amides; The olefin sulfonic acid root; Olefin sulfonic acids etc. contain heteroatomic loop section, or contain heteroatomic aliphatic portion, and wherein every kind can be optionally substituted; The exemplary heteroatomic part that contains comprises the optional assorted alkyl that replaces, comprise methoxyl methyl, ethoxymethyl etc., heterocyclic radical comprises piperidyl etc., alkyl azochlorosulfonate and aryl sulfonic acid root, comprise the methylsulphonic acid root, 4-chlorobenzenesulfonic acid root etc., alkoxyl group comprises methoxyl group, oxyethyl group etc., amino, comprise methylamino, dimethylamino etc., carbonyl derivative comprises alkyl and aryl carbonyl, alkyl amino-carbonyl, carbalkoxy etc., assorted thiazolinyl, comprise the alkenyl amino alkyl, alkene oxygen base alkyl, the alkylamino thiazolinyl, the alkoxyl group thiazolinyl, alkylidene amino alkyl etc., assorted allyl group, ester, amine, acid amides, phosphorus-oxygen and phosphorus-to-sulfur bonds; And comprise United States Patent (USP) 5,658,751 and the open WO 00/66664 of PCT described in contain heteroatomic part; Every open all passing through with reference to intactly making up in the present invention;

T=0 or 1;

Z is selected from 0 or 1 charge number;

R ³, R ⁹And R ¹⁰(comprise C from hydrogen and alkyl independently of one another _1-6Alkyl and C _2-6Alkyl) selects in;

N=0,1 or 2; And

Q is a heterocycle, and such as pyridine, pyrimidine, quinoline or purine, wherein every kind can be optionally substituted.

Here employed term " alkyl " typically refers to and comprises the 1 linear or optional ramose hydrocarbon moiety to about 12 carbon atoms, for example includes but not limited to methyl (Me), ethyl, propyl group, butyl, dodecyl, 4-ethyl pentyl group etc.

Here employed term " cycloalkyl " typically refers to and comprises the 3 linear or optional ramose hydrocarbon moieties to about 14 carbon atoms, at least its part forms one or two ring, for example include but not limited to cyclopropyl, cyclopentyl, cyclohexyl, 4-methylcyclohexyl, 2, the 3-Dimethylcyclohexyl, 3,5-Dimethylcyclohexyl ethyl etc.

Here employed term " aryl " typically refers to the aromatic nucleus part, for example includes but not limited to phenyl (Ph), naphthyl, furyl, thienyl, pyrryl, pyrazolyl, isoxazolyl, isothiazolyl, oxazolyl, thiazolyl, pyridyl, pyridazinyl, pyrimidyl, pyrazinyl, quinolyl, isoquinolyl, quinoxalinyl, quinazolyl etc.

Here employed term " optional replace " typically refers to that one or more hydrogen atoms replace substituent described here such as halogen with substituent is optional on the parent's group that comprises carbon, nitrogen, oxygen or sulphur atom; Hydroxyl; Amino; Thio group; Alkyl, cycloalkyl, haloalkyl, halogenated cycloalkyl; Alkoxyl group, cycloalkyloxy, halogenated alkoxy; Alkyl monosubstituted amino and dialkyl amido; Aminoalkyl; Alkyl monosubstituted amino alkyl and dialkyl aminoalkyl; Alkylthio; Alkyl, haloalkyl, cycloalkyl and aryl glycosyl; Alkyl, haloalkyl, cycloalkyl and fragrant carbonyl oxygen base; Alkyl, haloalkyl, cycloalkyl and aryl sulfonyl; And as the carboxy derivatives of carboxylic acid, ester and acid amides.Need recognize, comprise together with the substituting of hydrogen atom with the nearside of the hydrogen of vicinal may make a common volution or the condensed ring of forming of the substituent that substitutes these hydrogen respectively.

Need recognize, above-mentioned each term can make up with the relevant mode of chemistry and be used to represent other parts, for example aralkyl refers to as herein defined that aromatic yl group and alkyl as herein defined are connected to form and includes but not limited to phenmethyl, styroyl, picolyl, 3, the structure of 5-dimethoxy-methyl pyridin-4-yl etc.

Need recognize that cyanine dyes structure as described herein can comprise chiral centre.In these cases, except other has explanation, need understanding in the description of these cyanine dyes structures, to comprise all steric isomers.The mixture of diastereomer that such steric isomer comprises simple optically active isomer, racemic mixture and contains one or more stereoisomerism conformations of any relative quantity.

Need also to recognize that cyanine dyes structure as described herein can comprise geometric centre.In these cases, except other has explanation, need understanding in the description of cyanine dyes structure, to comprise all geometrical isomers.Such geometrical isomer comprises the cis of how much conformation forms of simple form or multiple blended, trans, E and Z isomer.Also need to understand, depend on the characteristic of the two keys that comprised in the cyanine dyes structure, such double bond isomer can depend on as conditions such as solvent composition, solvent polarity, ionic strength cis and trans between or conversion mutually between E and the Z conformation.

Further need recognize, when electric charge Z greater than 0 the time, may have several tautomers of compound in structural formula I, also comprise the mixture of such tautomer.For example, electric charge Z can be positioned at as on the described nitrogen-atoms of structural formula I in form, or is positioned on the carbon atom that forms two heterocyclic polyenoid hydrocarbon chains of connection, or selects as another kind of, and electric charge can be positioned on the heterocycle Q.The tautomer of the charging cpd of structural formula I can be by compound in structural formula I rearrange (as the following exemplary configurations) of two keys-singly-bound conformation be described.

Wherein

X, R ², R ³, R ⁹, R ¹⁰Define with Q such as structural formula I, and t=1, Z=1, and n=1.Cyanine dyes compound as described herein comprises the equilibrium mixture of any one or these tautomer in several possible tautomers.It will be appreciated that the location of described surface charge is subjected to

X, R ², R ³, R ⁹, R ¹⁰Influence with Q characteristic partly.Need understand further that the equilibrium mixture of dominant tautomer or tautomer may depend on such as conditions such as solvent composition, solvent polarity, ionic strength, prescriptions.It will be appreciated that term " resonance structure " also refers to these multiple electric charge location and similarly described above formula.

Also need to understand, carry in compound in structural formula I under the situation of net charge (such as when the Z=1, or under situation about existing on the compound in structural formula I as charged substituent such as ammonium group or sulfonic acid group), these compound in structural formula I are with counterion.Comprise any unit price, divalence or multivalence counterion here in the description of included cyanine dyes structure.Exemplary counterion comprises as electronegative counterions such as iodide, muriate, bromide, oxyhydroxide, oxide compound, acetate, trifluoracetic acid root, a phosphate radical, gen-diphosphate, triphosphate, and as the counterion of the positively charged of lithium, sodium, potassium, caesium, ammonium, many alkylammoniums etc.Such counterion may come from used synthetic method, purification scheme or other ion exchange processes.

As if character or the kind of believing counterion can not influence the functional of cyanine dyes described here.Need recognize, when dyestuff described here is dissolved in the solvent that is used for putting into practice PCR reaction mixture described here, method and composition or other media, the counterion of following can with other counterions exchanges of existing in solvent or other media.Additional counterion like this can be lyate ion, root, damping fluid and/or metal.

Need recognize R ²In fact group can be any parent compound (wherein t=Z=0) that is derived from structural formula I

And have formula R ²(wherein L is suitable leavings group, R to the compound of-L ²As top definition) between the group of nucleophilic reaction.For example, R ²Be optional alkyl, acyl group, aryl, sulfonic acid or the alkylsulfonyl group that replaces, wherein every kind can be optionally substituted.Exemplary leavings group L includes but not limited to the halogenide as muriate and bromide; acylate as acetate, formate, trifluoracetic acid root; as the sulfonate radical of methylsulphonic acid root, trifluoromethane sulfonic acid root and tolyl sulfonate radical, as sulfate radical of methylsulfate etc.In a kind of illustrative embodiment, Q is a kind of heterocycle, such as but not limited to:

R wherein ⁴, R ⁵, R ⁶, R ⁷, R ⁸, R ¹¹, R ¹², R ¹³And R ¹⁴Be independently to be selected from hydrogen, halogen, alkyl, cycloalkyl, assorted alkyl, Heterocyclylalkyl, thiazolinyl, polyene-based, alkynyl, carbene base, Ene alkynyl base, aryl, heteroaryl, alkoxyl group, alkylthio, dialkyl amido separately, wherein every kind can be optionally substituted.

In the illustrative embodiment of another kind, R ⁴, R ⁵, R ⁶, R ⁷, R ⁸, R ¹¹, R ¹², R ¹³And R ¹⁴One of be as United States Patent (USP) 5,658, contain heteroatomic part described in 751.In the illustrative embodiment of another kind, R ⁴, R ⁵, R ⁶, R ⁷, R ⁸, R ¹¹, R ¹², R ¹³And R ¹⁴One of be a kind of reactive group; described reactive group includes but not limited to halogen, hydroxyl,-oxyl, amine, carboxylic acid, halogenide, ethanol, acetaldehyde, mercaptan, alkyl and aryl mercaptan, alkyl and aryl sulfonyl, succinimide ester, ketone and isothiocyanic acid, and they can be used for by for example forming the core texture that C-C, acid amides, ether, thioether, disulfide linkage, ketone, thiocarbamide and azomethine (Schiff bases) are connected to described part dyestuff.In the illustrative embodiment of another kind, R ⁴, R ⁵, R ⁶, R ⁷, R ⁸, R ¹¹, R ¹², R ¹³And R ¹⁴One of be bridging dyestuff (BRIDGE-DYE) with following structural formula:

Wherein

X, R ², t, Z, R ³, R ⁹, R ¹⁰, Q and the definition of n as structural formula I is done, bridge is a kind of single covalent linkage, or linear or ramose, ring or heterocyclic, saturated or unsaturated covalent linkage, have 1-16 the non-hydrogen atom as carbon, nitrogen, phosphorus, oxygen and sulphur, described like this key comprises alkyl, ether, thioether, amine, ester or amido linkage; Singly-bound, two key, triple bond or fragrant C-C; Phosphorus-oxygen, phosphorus-sulphur, nitrogen-nitrogen or nitrogen-oxygen key; Or the arbitrary combination of fragrance or assorted aromatic gp.Need recognize that in some embodiment, this dimeric structure is symmetric around bridge, in other embodiment, this dimeric structure is asymmetric around bridge, wherein for example

X, R ², t, Z, R ³, R ⁹, R ¹⁰With any among the n be independent separately the selection in each side of bridge in each case.

The exemplary dyes of using among the present invention also comprises the cyanine dyes of the structural formula I with pyridine or pyrimidine core texture, and wherein X represents oxygen or sulphur;

The optional molten benzene that replaces of part representative, the optional molten naphthalene that replaces, the optional molten pyridine that replaces, the optional molten pyrimidine that replaces, the optional molten quinoline that replaces etc.; N=0 or 1; T=0 or 1; R ²Be alkyl, as methyl and ethyl, the optional aryl that replaces, as phenyl or tolyl, the olefin sulfonic acid root, as propyl group alkene sulfonic acid, or alkyl sulphonyl, as CH ₃(CH ₂) _mSO ₂, wherein m is 0,1,2 or 3; And Q is the heterocycle that is selected from following structure:

Wherein

R ⁴Be hydrogen, alkoxyl group, comprise methoxyl group, oxyethyl group, propoxy-etc.; Alkylthio comprises methylthio group, ethylmercapto group etc.; Heterocyclylalkyl comprises the optional piperidyl that replaces, pyrrolidyl, piperazinyl etc.; Or comprise the Heterocyclylalkyl of charged group, comprise 4,4-lupetazin-1-base etc.; Or reactive group, comprise halogen, hydroxyl, alkoxyl group, thio group, alkyl and artyl sulfo, alkyl and aryl sulfonyl, amino, formyl radical, alkyl and aryl carbonyl, carboxy derivatives etc.;

R ⁵Be C _1-6Alkyl comprises methyl, ethyl, butyl, sec-butyl, isobutyl-etc.; The optional phenyl that replaces; Or (CH ₂) ₃N ⁺(ME) ₃And

R ⁶, R ⁷And R ⁸Be independently hydrogen or methyl separately.

Exemplary dyes used herein also comprises the cyanine dyes of the structural formula I with pyridine or pyrimidine core texture, and wherein X is oxygen or sulphur;

The part representative forms the molten benzene of the optional replacement of optional benzoxazole that replaces or benzothiazole ring, or forms the molten naphthalene of the optional replacement of optional Nai Bing oxazole that replaces or aphthothiazoles ring; N=0 or 1; T=0 or 1; R ²Be alkyl, as methyl, aryl, as phenyl or tolyl, the olefin sulfonic acid root, as propene sulfonic acid, or alkyl sulphonyl, as CH ₃(CH ₂) _mSO ₂, wherein m is 0,1,2 or 3; And Q is 4-pyridine or 4-pyrimidine heterocyclic.

Here employed exemplary dyes is also included within the cyanine dyes with quinoline nuclei core structure useful in PCR mixture, method and the composition described here, and describes with structural formula II usually:

Structural formula II

Wherein

The part representative is chosen the monocycle that replaces wantonly or is encircled aromatic nucleus or nitrogenous hetero-aromatic ring more;

X is oxygen, sulphur or is selected from C (CH ₃) ₂And NR ¹Group, R wherein ¹Be hydrogen or C _1-6Alkyl;

R ²Be alkyl, comprise C _1-6Alkyl and C _2-6Alkyl, cycloalkyl comprises C _3-8Cycloalkyl, aryl, aralkyl, the olefin sulfonic acid root contains heteroatomic loop section, or contains heteroatomic aliphatic portion, and wherein every kind can be optionally substituted;

T=0 or 1;

Z is selected from 0 or 1 charge number;

R ³, R ⁹And R ¹⁰Independently be selected from hydrogen separately and comprise C _1-6The alkyl of alkyl;

N=0,1 or 2; And

R ⁴, R ⁵, R ⁸, R ¹¹, R ¹², R ¹³And R ¹⁴By the description of here structural formula I being done, if R ⁴Be molecular weight less than about 115, or exemplarily molecular weight less than 105 part;

The exemplary dyes of using among the present invention also comprises the cyanine dyes of structural formula II, wherein

The optional molten benzene that replaces of part representative, thus benzoxazole or benzothiazole ring formed; X is oxygen or sulphur; N=0 or 1; T=0 or 1; R ²It is methyl;

R ⁴Be hydrogen, C _1-6Alkyl comprises methyl, or the optional phenyl that replaces;

R ⁵Be C _1-6Alkyl comprises methyl, or the optional phenyl that replaces;

R ⁸Be hydrogen, and

R ¹¹, R ¹², R ¹³And R ¹⁴Be hydrogen or alkoxyl group, comprise methoxyl group.

In other embodiments, employed dyestuff also exemplarily comprises the cyanine fuel of structural formula II among the present invention, wherein

The optional heterocycle that replaces of part representative, comprise the 1-picoline and and 3-bromo-1-pyrido methyl; X is oxygen or sulphur; N=0 or 1; T=Z=0;

R ⁴Be hydrogen or C _1-6Alkyl comprises methyl;

R ⁵Be C _1-6Alkyl comprises methyl, and optional phenyl or the cycloalkyl that replaces comprises having charged group such as group-(CH ₂) ₃N ⁺(ME) ₃Cycloalkyl;

R ⁸Be hydrogen; And

R ¹¹, R ¹², R ¹³And R ¹⁴Be hydrogen, alkyl comprises methyl, or alkoxyl group, comprises methoxyl group.

In another embodiment, two kinds of compounds with structural formula I merge to form dimer.Described two kinds of compounds are by substituting the substituent R as defined above on the every kind of compound that is present in structural formula I with single divalence connector ⁴, R ⁵, R ⁶, R ⁷, R ⁸, R ¹¹, R ¹², R ¹³And R ¹⁴One of interconnection.For example, two kinds of compounds of combinatorial construction formula I wherein are present in two kinds of R on described two kinds of compound in structural formula I to form dimer ⁵Substituent substitutes with single divalence connector.Need recognize, consider the symmetry and the asymmetric dimer of structural formula I compound here.Under the asymmetric dimeric situation of compound in structural formula I, need understand, by forming dimer and can produce so asymmetric by having compound in structural formula I different replacement modes or that have different heterocycle Q.Further, the dimer that forms by the different substituents with the compound of described divalence connector alternative structure formula I can produce so asymmetric, for example substitutes R on first kind of structural formula I compound with described divalence connector ⁵And the R on alternative second kind of structural formula I compound ⁸

In another embodiment, two kinds of compounds with structural formula II merge to form dimer.Described two kinds of compounds are by substituting the substituent R as defined above on the every kind of compound that is present in structural formula II with single divalence connector ⁴, R ⁵, R ⁸, R ¹¹, R ¹², R ¹³And R ¹⁴One of interconnection.For example, two kinds of compounds of combinatorial construction formula II to be to form dimer, wherein are present in two kinds of R on the compound of described two kinds of structural formula II ⁵Substituent substitutes with single divalence connector.Need recognize, consider the symmetry and the asymmetric dimer of structural formula II compound here.Under the asymmetric dimeric situation of the compound of structural formula II, need understand, so asymmetric by producing by compound formation dimer with structural formula II different replacement modes or that have different heterocycle Q.Further, the dimer that forms by the different substituents with the compound of described divalence connector alternative structure formula II can produce so asymmetric, for example substitutes R on first kind of structural formula II compound with described divalence connector ⁵And the R on alternative second kind of structural formula II compound ⁸

Also can represent by the dimer cyanine dyes structure that compound in structural formula I forms with structural formula II I:

Structural formula II I

Wherein

With

Part is represented respectively and independently is selected from the optional fusion monocycle that replaces or encircles aromatic nucleus or nitrogenous hetero-aromatic ring more;

X and X ' independently are selected from oxygen, sulphur, selenium, tellurium separately or are selected from C (CH ₃) ₂, NR ¹Or NR ¹' group, NR wherein ¹And NR ¹' be independently hydrogen or C separately _1-6Alkyl;

R ²And R ²' be independently to be selected from alkyl separately, comprise C _1-6Alkyl, cycloalkyl comprises C _3-8Cycloalkyl, aryl, aralkyl comprises aryl (C _1-2Alkyl), contain heteroatomic loop section, or contain heteroatomic aliphatic portion, wherein every kind can be optionally substituted;

T=0 or 1;

T '=0 or 1;

Each independently is selected from 0 or 1 charge number naturally Z and Z ';

R ³, R ⁹, R ¹⁰, R ³', R ⁹' and R ¹⁰' independently be selected from hydrogen and alkyl separately, comprise C _1-6Alkyl;

N=0,1 or 2;

N '=0,1 or 2;

Bridge is the divalence connector that constitutes to about 30 divalent units by 2, and described divalent unit is selected from thiazolinyl, assorted thiazolinyl, alkane two amidos (alkylamindiyl), alkyl-alkyl two ammoniums (alkylalkylammoniumdiyl) etc., as (CH ₂) _P, (CH ₂) _pN ⁺Me ₂(CH ₂) _q, (CH ₂) _pN ⁺Me ₂(CH ₂) _qN ⁺Me ₂(CH ₂) _rDeng, wherein p, q and r independently are selected from 1,2 and 3 separately; And

Q and Q ' are heterocycles, independently are selected from down array structure separately:

R wherein ⁴, R ⁵, R ⁶, R ⁷, R ⁸, R ¹¹, R ¹², R ¹³And R ¹⁴Independently be selected from hydrogen, halogen, alkyl, cycloalkyl, assorted alkyl, Heterocyclylalkyl, thiazolinyl, polyene-based, alkynyl, carbene base, Ene alkynyl base, aryl, heteroaryl, cycloalkyl in the compound of every kind of structural formula II I, wherein every kind of group can be optionally substituted.

Exemplary cyanine dyes useful in PCR reaction mixture of the present invention, method and composition also includes but not limited to: LightCycler Green, PO-PRO ^TM-1, BO-PRO ^TM-1,

43,

44,

45,

Blue, POPO ^TM-1, POPO ^TM-3, BOBO ^TM-1, BOBO ^TM-3, and other dyestuffs with general formula I V:

Structural formula IVa structural formula IVb

And dyestuff G5, the H5, D6, E6, P6, R6, Y6, Z6 and the D8 that are showed among the embodiment 14, and other dyestuffs with general formula V:

Structural formula Va structural formula Vb

Wherein n is 0,1 or 2; R ²Be alkyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl, monoalkyl and dialkylaminoalkyl group, trialkyl ammonium alkyl, thiazolinyl carboxylate radical, thiazolinyl carboxylic acid amides, olefin sulfonic acid root etc.; R ⁵Be alkyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl, monoalkyl and dialkylaminoalkyl group, trialkyl ammonium alkyl, thiazolinyl carboxylate radical, thiazolinyl carboxylic acid amides, olefin sulfonic acid root, the optional phenyl that replaces etc.; X is oxygen or sulphur; A, A ', the optional substituent of representing one or more independently to select separately with B, as alkyl, halogen, amino, haloalkyl, alkoxyl group, halogenated alkoxy, alkyl and aryl sulfonyl, halogenated alkyl sulfonyl, alkyl and artyl sulfo, formyl radical, alkyl and aryl carbonyl, carboxy derivatives, monoalkyl and dialkyl amido, trialkyl ammonium, dialkylaminoalkyl group, the trialkyl ammonium alkyl, or comprise that tetramethyleneimine also, piperidines also, (wherein every kind of group can be used alkyl to piperazine heterocycle also, amino, monoalkyl or dialkyl aminoalkyl, the trialkyl ammonium alkyl replaces, or available alkyl is optional quaternized on nitrogen) etc.; Bridge is to have structural formula (CH ₂) _pN ⁺Me ₂(CH ₂) _qDivalence connector (wherein p and q equal 2 or 3 independently), comprise divalence connector (CH ₂) ₃N ⁺Me ₂(CH ₂) ₃Need to understand, when these dyestuffs had net charge, they were with one or more counterions, as comprised the balance anion of halogenide, alkane acid group, phosphate radical etc., and the balance cation that comprises lithium, sodium, potassium, caesium, ammonium etc.

Here employed other exemplary dyes include but not limited to YO-

-1, TO-

-1, 11,

13,

15, 16,

20,

23, TOTO ^TM-3,

-3 (Molecular Probes, Inc.),

(Cambrex Bio ScienceRockland Inc., Rockland, ME), Thiazole Orange (Aldrich), and other dyestuffs with general formula VI:

Structural formula VIa structural formula VIb

Wherein n is 0,1 or 2; R ²Be alkyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl, monoalkyl and dialkylaminoalkyl group, trialkyl ammonium alkyl, thiazolinyl carboxylate radical, thiazolinyl carboxylic acid amides, olefin sulfonic acid root etc.; R ⁵Be alkyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl, monoalkyl and dialkylaminoalkyl group trialkyl ammonium alkyl, thiazolinyl carboxylate radical, thiazolinyl carboxylic acid amides, olefin sulfonic acid root, the optional phenyl that replaces etc.; X is oxygen or sulphur; A, A ' and B represent one or more optional substituents of independently selecting separately, as alkyl, halogen, amino, monoalkyl and dialkyl amido, tetramethyleneimine also, piperidines also, piperazine also, phenyl, hydroxyl, alkoxyl group, thio group and alkylthio, wherein every kind of group can be used replacements such as alkyl, amino, monoalkyl or dialkyl aminoalkyl, trialkyl ammonium alkyl; Bridge is to have structural formula (CH ₂) _pN ⁺Me ₂(CH ₂) _qThe divalence connector, wherein p and q equal 2 or 3 independently, comprise divalence connector (CH ₂) ₃N ⁺Me ₂(CH ₂) ₃Need to understand, when these dyestuffs had net charge, they were with one or more counterions, as comprised the balance anion of halogenide, alkane acid group, phosphate radical etc., and the balance cation that comprises lithium, sodium, potassium, caesium, ammonium etc.

Further, table 1 (providing among the embodiment 13 below) represented in the PCR process or normally used afterwards several double-stranded DNA dyestuff and before also be not used for the comparison of the multiple dyestuff of pcr analysis.Initial results shows, for the detection of heteroduplex molecule, and LC Green, PO-PRO ^TM-1, JO-PRO ^TM-1, BO-PRO ^TM-1, G5, H5, D6, P6, Y6 and D8 are dyestuffs likely.These dyestuffs have several surprising character.The first, under 50% saturation ratio, these dyestuffs can obviously not suppress PCR.In fact, with three kinds in these dyestuffs, quite compatible with PCR near 100% saturated level.The second, though some in these dyestuffs are luminous between blue light region, they can use in the fluorescein passage of multiple present available instrument.Can further improve their sensitivity in the use that quantitative or qualitative amplification is analyzed for the excitation/emission wide spectrum that mates these dyestuffs better to the adjustment of optics.

Need to understand, above cyanine dyes is exemplary, and other cyanine dyes also can use in described method.

Such as, but not limited to

Green I,

Green,

14,

21,

24,

25, TOTO ^TM-1 He

Some of-1 are not proved to be the stopped pipe detection that can be used for heteroduplex molecule detection or multiple product based on the asymmetric cyanine of quinoline.When described dyestuff is a kind of monomer of the cyanine based on quinoline, (be equivalent to R on the carbon near the nitrogen of quinoline ring ⁴The position) replacement of large volume may disturb described dyestuff to bring into play the ability of function in the method for the invention.Large volume replaces and to be meant, for example, contains heteroatoms aliphatics or aromatic series part with molecular weight greater than about 105 side chain aliphatic portion replacement long chain branches.Yet this restriction is not suitable for above-mentioned any pyridine or pyrimidine cyanine.Under based on the dimeric situation of the cyanine of quinoline, as it seems by the distance between the defined right and left rings of the following divalence fragment system also determine functional.

Functional can be as described in the embodiment 13-14 here, detect by heteroduplex molecule and to be determined.Such as

Other dyestuffs that can be used for PCR monitoring in real time of describing before Gold, Pico Green and the ethidium bromide etc. also have been proved to be invalid in the heteroduplex molecule of stopped pipe PCR system detects.

The dyestuff that uses among the present invention can use in the SNP methods of genotyping based on dyestuff, two kinds of unlabelled Oligonucleolide primers of these needs and at every kind of genotypic reaction tank of SNP, and do not require PCR in real time.Use a kind of double-stranded DNA dyestuff, pass through to change the existence of the heteroduplex molecule of melting curve shape after increasing like this and differentiate heterozygote.Different homozygote genotype are by its Tm difference, or select as another kind of, mix with unknown sample and seek heteroduplex molecule and distinguish by known homozygote DNA sample.For example, because can use very short amplicon, preferably directly and the amplicon that joins of SNP flank, the PCR design of primers has obtained very big simplification.The amplicon of such weak point is amplification very effectively simultaneously, reduces the risk of other targets of amplification, and makes very fast that thermal cycling becomes possibility.

The PCR design of primers is not an accurate science, and frequent trial and mistake are inevitable.Though the criterion of some PCR design of primers is widely accepted, the exactness of these criterions does not also obtain check.Because different genotype is bigger to the influence of melting curve during with short amplicon, preferred short amplicon (≤100bp), and best usually be the shortest possible amplicon (≤50bp).Therefore, in order to be designed for primer, for example from being close to every flank primer in SNP site with double-stranded DNA dyestuff gene type.Like this, amplicon length will be that the length of primer 1 adds that the length of primer 2 adds the length (length of a SNP is 1) in the zone that need detect.For effective amplification, the melting temperature(Tm) of two primers (Tm) should be basic identical.The suitable Tm of primer can be 50-70 ℃.For example, the primer of the highest Tm value can make the fastest thermal cycling become possibility, and the lower cheap usually and the shortest amplicon of generation of primer of Tm value, this causes bigger gene type difference.Usually use the primer of length between 12 to 30 bases.For example, every kind of primer makes up the Tm value that approaches to wish most up to the Tm value of calculating outward from SNP.The method of calculating the Tm value is to know (Clin.Chem.2001 for example in this area; 47:1956-61).Usually, when the Tm value was mated as far as possible, primer length should be different.For example, the primer length that uses in the snp analysis (Fig. 1) of factor V is 17 and 24 bases, and both have the Tm value of mating near 62 ℃ calculating.

Because the amplicon for such weak point needs primer extension hardly, the thermal circulation parameters of amplification can be very short.After the initial sex change of genomic dna before the thermal cycling, do not need to keep sex change and annealing temperature, the extension time can be 10 seconds or still less.Even the extension time of programming can be reduced to 0, so that circulating within 20 seconds, each carries out.In addition, can use 1 second extension time.Because amplicon is so short, do not need a large amount of polysaccharase (can use＜per 10 microlitres of 0.6 unit).

Thereby, according to the present invention, can carry out the SNP gene type according to following illustrative steps:

1. select target Tm value and 3 ' terminal from every kind of primer in next-door neighbour SNP site.Optional, thus primer can be removed from the SNP site a little to avoid 3 ' the complementary risk that forms primer dimer that reduces between the primer.

2. outwards design every kind of primer until the Tm value of calculating as far as possible near target Tm value.

3. under the condition that the double-stranded DNA dyestuff that has PCR reagent and allow heteroduplex molecule to detect exists, sample is carried out rapid thermal cycles.

4. is at least-0.1 ℃/second by speed after the sex change, preferably at least-2 ℃ of/second, ℃/second quick cooling forms heteroduplex molecule most preferably at least-5.

5. with the heating of 0.1-0.5 ℃/second speed and obtain melting curve.

6. if described amplification failure moves 1 base and repeats all steps up to achieving success outside the 3 ' end with a primer.

In an illustrative example, all heterozygotes can be detected (Fig. 4) to the influence of melting curve by heteroduplex molecule.In addition, 4 kinds in 6 kinds of homozygote difference (A to C, A to G, C to T and G to T) are easy to move by Tm and distinguish (Fig. 4, arrow).Yet, in order to distinguish A, may need high resolving power to unwind, and, can not distinguish C to the G homozygote even if use high resolving power to unwind in some cases to the T homozygote.Under the situation that homozygote is difficult to distinguish, the genotypic sample of known homozygote can be mixed with described unknown gene type with roughly suitable amount before the amplification or after the amplification.To this mixture increase (if in the past amplification), sex change and unwinding.If described genotype is identical, the melting curve of mixture should be identical with the genotypic melting curve of known homozygote.If described genotype difference can produce heteroduplex molecule and differentiated by the change of melting curve shape.For example, when the known array varient is carried out gene type, can use little amplicon.When the unknown varient of scanning, can preferably big amplicon.

Asymmetric cyanine dyes can be prepared by usual method, and this method is connected the indoles of molecule part and pyridine (or quinoline, pyrimidine, purine) part by one or more " C (R)=" group.As United States Patent (USP) 5,436,134 and the reference of quoting described in, the quantity of " C (R)=" group is by the concrete synthetic agent decision of using in synthetic.At single methyl dyestuff (R=H as LC Green; n=0) in synthetic; used a kind of combination of reagent; wherein the methine carbon atom is by generating as the reactive leavings group outside the A on the indoles salt of methyl or the B on the pyridinium salt and A and the B; described leavings group is methylthio group, methyl sulphonyl or chloro normally, but can provide enough reactivities to finish any leavings group of reaction.A kind of method that can prepare LC Green and other similar dyestuffs is as follows:

Parent material compound 1 was prepared by 4-methyl-2-pyridone (Aldrich) is heated to reflux with copper powder, salt of wormwood and phenyl-iodide in 48 hours.Reaction is cooled to room temperature, between water and ethyl acetate, distributes mutually, filter, and organic layer is dry on sal epsom.Crude product is purifying on silicagel column, with 1: 1 ethyl acetate/hexane wash-out with results compound 1.

Another kind of parent material, compound 2, be as a kind of salt compounded of iodine add 2-(methylthio group) benzothiazole by the methyl-iodide in DMF and in the sealing test tube 150 ℃ of heating 1 hour be prepared with acquisition compound 2.

The mixture heating up of the DMF of catalytic amount is to refluxing 24 hours in compound 1, Phosphorus Oxychloride and the methylene dichloride.Mixture is cooled to room temperature and adds the methylene dichloride of another part volume, adds compound 2 and 1 normal triethylamine subsequently.Stirred 6 hours under the mixture room temperature.Use the mixture wash-out of ethyl acetate/chloroform/methanol to carry out purifying by the filtering separation solid and with silicagel column.The compound of purifying heavily is dissolved in methyl alcohol subsequently and adds excessive sodium iodide aqueous solution.Compound 3 passes through filtering separation and vacuum-drying with the form of salt compounded of iodine.

Compound 3 is subsequently with 1, and the 1-methylpiperazine in the 2-ethylene dichloride mixes and 55 ℃ of heating 2 hours.The product (compound 4) that obtains is quaternized by adding excessive methyl-iodide and Proton Sponge (Aldrich) subsequently, the LightCycler Green (compound 5) of expection results two iodate salt forms.

Embodiment 1

The PCR scheme

Mark with unlabelled oligonucleotide from IT Biochem (Salt Lake City, UT), QiagenOperon (Alameda, CA) or Synthegen (Houston TX) locates to obtain.Except other had explanation, the PCR of 10 microlitre volumes existed

(Roche Applied Systems, Indianapolis IN) upward carry out with 20 ℃/second procedural variation.Except other had explanation, amplification mixture had comprised that 50 nanograms are as the genomic dna of template, every kind of dNTP each 200 μ M, 3mM MgCl ₂, 100mM 2-amino-2-methyl-1, ammediol, pH 8.8,0.04 units/microlitre Taq polysaccharase (Roche), 500 mcg/ml BSA and every kind of each 0.5 μ M of primer.The human genome DNA of gene type is from former research (Gundry CN etc., Genetic Testing, 1999; 3:365-70; Herrmann M etc., Clin Chem2000; 46:425-8) or from the Coriell cell bank (Camden, NJ).Except other has explanation, comprise the LightCycler Green of 10 μ M in the PCR reaction.Using

Under the situation of Green I as indicator, used Molecular Probes storage liquid 1: 10,000 finally dilution.Before PCR, add dyestuff, increase, and

Go up or monitor the transformation of unwinding of amplicon by the high resolving power melting analysis.Distinguish different homozygotes by amplicon melting temperature(Tm) (Tm).By the low temperature that can widen total heteroduplex molecule between the limited proportionality that the unwinds discriminating heterozygote that unwinds.Melting analysis needs about 1 minute, and does not need the sampling process behind the PCR.

For study LC Green,

The sensitivity of Green I and other double-stranded DNA combination dyes, analyzed factor V Leiden, cystic fibrosis (F508del, F508C, I507del, I506V) and the polymorphism in HTR2A (T102C) gene.In addition, used engineering plasmid with the possible single sequence change of comprehensive institute.The preferably quick cooling by denatured products in the melting analysis process of the heteroduplex molecule that produces by the heterozygote DNA cloning (at least-2 ℃/second), detect by rapid heating (0.2 to 0.4 ℃/second) subsequently.It is to have the more transformation of unwinding of wide region that all heterozygotes are different from homozygous.Different homozygotes often can be distinguished by its Tm value.Having A can only distinguish by the high resolving power melting analysis or by the homozygote mixing to the homozygote of T sequence change.Homozygote G does not carry out homozygote blended words to the C sequence change, even use Analytical high resolution repeatedly to be distinguished.Described amplicon length changes between 44 to 331bp.

Though LC Green has been provided among the embodiment that provides here, should be appreciated that, can use corresponding to other dyestuffs of the present invention.

Embodiment 2

Curve analysis

Exist immediately after the circulation

Go up or separate in high resolving power subsequently that (Salt Lake City UT) carries out curve analysis for HR-1, IdahoTechnology on the chain apparatus.Yet, need understand curve analysis can not have the amplification situation under carry out.Using

The time, sample at first is heated to 94 ℃, is cooled to 60 ℃ with-20 ℃/second sequencing settings, unwinds with 0.2 ℃/second then and obtains fluorescence continuously.Unwind as for high resolving power, except other had explanation, sample at first existed

Middle amplification exists subsequently

In instantaneously heating to 94 ℃ and cooling fast (program setting-20 ℃/second) to 40 ℃.Except other has explanation, then will

Each one of kapillary is transferred to high resolution device and with 0.3 ℃ of/second heating.HR-1 surrounds one with aluminium matter cylinder Single sampling unit capillaceous.System is heated by joule by the spiral groove around the cylinder outside.Sample temperature is with placing the thermocouple monitoring of cylindrical inside equally and converting 16 bit digital signal to.The internal illumination of the capillary end of fluorescence by being positioned at cylindrical base monitor (Wittwer CT etc., BioTechniques 1997; 22:176-81) also convert 16 bit signals equally to.Obtain about 50 data points to every ℃.

In some cases, after the PCR before obtaining melting curve the product unchangeability its benefit is arranged.For example, when target is the quantity somatotype of tumor-necrosis factor glycoproteins (for example STRs and VNTRs), stops on the extension step of amplification in can the index of Response phase process before plateau, and carry out melting analysis subsequently.Like this, can analyze homoduplex molecule extension products.In the tumor-necrosis factor glycoproteins somatotype, particularly because many different heteroduplex molecule products may be arranged by the difference alignment of tumor-necrosis factor glycoproteins to be formed, so the homoduplex molecular product can provide more information than heteroduplex molecule product.In some cases, both obtained homoduplex molecule melting curve (not sex change premenstruum), it may be useful obtaining heteroduplex molecule melting curve (through sex change and form all possible double-stranded combination) again.Use same sample as " homoduplex contrast ", the difference between these two kinds of melting curves has provided measuring of the heteroduplex molecule degree that can form.

Analyze with the customized software of writing among the Lab View with the high resolving power data of unwinding.Fluorescence-hygrogram by at first define every kind of sample unwind change before and afterwards linear baseline between 0 to 100%, carry out stdn.In every kind of sample, the fluorescence of each acquisition calculates as fluorescence per-cent between this acquisition temperature next top and the bottom baseline.In some cases, the derivative melting profile calculates (" digital processing in the C language " (Numerical recipes in C) of people such as Press WH chief editor by the Savitsky-Golay polynomial expression to a point, second edition, New York:Cambridge University Press, 1992:650-5).Savitsky-Golay analyzes and uses 2 order polynomials and comprise the data window of being had a few within 1 ℃ of interval.By using the non-linear least square Return Law, peak area and melting temperature(Tm) have been obtained to be fit to multiple Gaussian function.In some cases, the X-axis of every standardized melting curve is moved so that figure is overlapping between certain phosphor region.This " temperature moves " proofreaied and correct any trickle operating temperature variation and improved the ability that heterozygote and homozygote are distinguished.Difference between the genotype also can be by amplifying in the mapping of the fluorescence difference between the genotype under each temperature.

Embodiment 3

Use the single nucleotide polymorphism gene type of LightCycler Green:

Factor V Leiden mutator gene somatotype

By length is the amplicon that 18 and 24 bases, the primer that is close to both sides, factor VLeiden mutational site form 43bp.Article two, primer all has 62 ℃ estimation Tm value.Sample uses following scheme circulation 35 times: 94 ℃ do not keep, 60 ℃ do not keep and 72 ℃ kept 10 seconds.After the amplification, sample exists

Interior instantaneously heating to 94 ℃, to 60 ℃, the PCR product unwinds with 0.2 ℃/second and obtains continuous fluorescence in cooling fast (program setting-20 ℃/second).

Shown among Fig. 1 from the derivative melting curve of the different genotype amplification PCR products in factor V gene Leiden site.The fluorescence monitoring that LightCycler Green is used for unwinding between two strands and the single stranded product and changes.The Leiden sudden change is positioned at 19 base places of amplicon one end.Shown from 10 homozygote wild-types, 2 heterozygotes and 1 result that homozygote Leiden genotype obtains.The amplicon melting temperature(Tm) of homozygote mutant is than low about 1 ℃ of homozygote wild-type melting temperature(Tm).Form owing to heteroduplex molecule, the heterozygote sample shows the transformation of unwinding of secondary a, low temperature.Use

The similar experiment of Green I does not detect the secondary transformation (data are unexposed) of unwinding in this heterozygote.

Last use heterozygote factor VLeiden DNA has studied the influence of speed of cooling and rate of heating.In order to study the influence of speed of cooling, the sample that increases as mentioned above is heated to 85 ℃, is cooled to 60 ℃ with-20 ,-2 ,-0.5 or-0.1 ℃/second speed from 85 ℃ subsequently, and the constant rate of heating by 0.2 ℃/second is to obtain melting curve afterwards.Cooling is essential (Fig. 2) for forming tangible heteroduplex molecule fast.When speed of cooling is-0.1 ℃/second or when lower, do not observe heteroduplex molecule.When the kapillary sample when boiling water is transferred to the frozen water fast, the heteroduplex molecule that maximum takes place forms (Fig. 2).Employing exists

Last cooling, heteroduplex molecule be formed on faster than-5 ℃/second speed is set the time it seems and reach maintenance level (Fig. 2).Yet the measurement of actual sample temperature shows that when speed was set faster than-5 ℃/second, speed of cooling only had trickle raising: when device was set to-20 ℃ of/second coolings, actual speed was about-6 ℃/second.

Cool off by the speed that is provided with, unwind with 0.05,0.1,0.3 or 0.5 ℃/second subsequently and studied the influence of rate of heating with-20 ℃/second.Consistent with higher rate of heating, the relative percentage of observed heteroduplex molecule higher (Fig. 3).Along with speed improves, apparent Tm value is also moved to higher temperature, and separates chain process and depart from (Gundry CN etc., Genetic Testing, 1999 from equilibrium state to a greater degree; 3:365-70).

Embodiment 4

Use the systematic study of plasmid to the SNP gene type

For the melting curve gene type to all possible single sequence change carries out systematic study, used engineering plasmid.Described plasmid (DNA Toolbox, Cambrex Bio Science Rockland Inc.) in 40%GC content, on definition position, comprised A, C, G or T (Highsmith WE etc., Electrophoresis 1999; 20:1186-94).Tm is 62 ± 1 ℃ a primer next-door neighbour pleomorphism site.Use 10 ⁵The dna profiling of copy and adopt 94 ℃ do not keep, 60 ℃ do not keep, 35 circulations that 75 ℃ kept 5 seconds carry out PCR.

Be used for melting analysis.

Fig. 4 explanation can be distinguished with the homoduplex molecule by all possible heteroduplex molecule that single base polymorphisms forms.In each case, the existence of heteroduplex molecule causes forming low temperature shoulder form on the derivative melting profile.Divide the period of the day from 11 p.m. to 1 a.m when sample includes only the homoduplex that is formed by the homozygote amplification, do not have low temperature shoulder form.And AA or TT homozygote are obviously distinguished by its melting temperature(Tm) and CC or GG homozygote.By these " low resolution " figures ( Last acquisition) can not determine whether all heterozygotes can be distinguished mutually, or AA can not distinguish with TT, CC can distinguish with GG.Yet high-resolution data (unexposed) shows that AA can distinguish with TT, and the overwhelming majority (if not all) heterozygote can be distinguished.The homozygous stability of CC and GG be it seems closely similar, and is not difficult to distinguish any difference by the homozygote mixing on existing instrument.

Embodiment 5

Adopt labeled primer, LightCycler Green or

The gene type of the cystic fibrosis gene of Green I

Used among the PCR KlenTaql polysaccharase (0.04 unit/microlitre, AB Peptides, St.Louis, MO), 88 nanogram TaqStart antibody (ClonTech, Palo Alto, CA) and 50mM Tris, pH 8.3, rather than Taq polysaccharase and 2-amino-2-methyl-1, ammediol.Use the increased fragment of a 44bp of following primer: GGCACCATTAAAGAAAATAT (SEQ ID NO:1) and TCATCATAGGAAACACCA (SEQ ID NO:2).Article one, primer or carried out 5 ' mark with Oregon Green, or be reflected at

Carry out under the condition that Green I or LightCycler Green exist.Primer is adjacent with the mutantional hotspot that comprises F508del, I507del and F508C varient.PCR carries out through 40 circulations of 85 ℃ and 58 ℃ (keeping 0 second).

6 samples have been monitored in the last melting curve procurement process.

Fig. 5 B-D has shown the derivative melting curve by different genotype amplification PCR products on the cystic fibrosis gene I507/F508 zone.PCR product length is 41 or 44 bases (Fig. 5 A).5 ' labeled primer (Fig. 5 B), LightCyeler Green (Fig. 5 C) or

The fluorescence monitoring that Green I (Fig. 5 D) is used for unwinding between strand and the double-stranded product and changes.Shown by two kinds of homozygotes and three kinds of results that the heterozygote genotype obtains.

The double-stranded stability of different genotype is followed Theoretical Calculation (vonAhsen N etc., Clin Chem 2001; 47:1956-61), F508del～I507del＜wild-type＜F508C wherein.Except F508del and I507del, genotype can be distinguished by the Tm value that they mainly change.

On when unwinding, the standard deviation that repeats the Tm of wild-type samples for 10 times is 0.12 ℃.When unwinding on high resolution device, the standard deviation of the Tm of identical 10 samples is 0.04 ℃.

When the heterozygote sample when the pcr amplification, expect two kinds of homoduplex molecules and two kinds of heteroduplex molecules (Nataraj AJ etc., Electrophoresis 1999; 20:1177-85).Yet, when

When Green I is used as fluorescent indicator, can only see the single peak that unwinds (Fig. 5 D) for every kind of genotype.On the contrary, when applying marking primer or LightCycler Green under the same conditions, two obviously peaks (Fig. 5 B and 5C) of definition have appearred.A kind of or whole transformation of unwinding of two kinds of heteroduplex molecule products is probably represented always less than the pyritous peak in the low temperature peak.Erect image can be expectable, and the heterozygote (F508del and I507del) of 3bp disappearance has caused the more unsettled heteroduplex molecule peak, heteroduplex molecule peak than single sequence change (F508C).The main peak position of F508C heterozygote is in the temperature higher than wild-type, and this has reflected more high stability (Gundry CN etc., Genetic Testing, 1999 of T to the G conversion; 3:365-70).

Embodiment 6

Adopt the sudden change scanning of LC Green

Studied the single nucleotide polymorphism of HTR2A.Carry out PCR according to description with KlenTaq, TaqStart and Tris to the cystic fibrosis site.The 331bp fragment of seretonine receptor 5 2A (HTR2A) gene has comprised total polymorphism (T102C) (Lipsky RH etc., the Clin Chem 2001 of exons 1 inside; 47:635-44).Be reflected at 95 ℃ do not keep, 62 ℃ keep keeping in 2 seconds and 74 ℃ between 20 seconds circulation 40 times.Obtained the high resolving power melting curve.

Fig. 6 shows that LightCycler Green can be used to the scanning to sequence variations.Do not need to know the position of sequence variations in other words.The existence of any variation can detect a big amplicon inside.As shown in Figure 6, three single nucleotide polymorphism genotype of all in the HTR2A gene (homozygote T, homozygote C and heterozygote T/C) can be distinguished in the amplicon of 331bp.The ability of the tolerance range of melting curve and differentiation different genotype depends on the temperature and the fluorescence resolving power of device.

Embodiment 7

The curve analysis of the big tick marks of DNA:

The comparison of Green I and LightCycler Green

100 nanograms contain the big tick marks of DNA (low-molecular-weight dna mark, Gibco BRL) of 6 kinds of different sorts double-stranded DNAs at 3mM MgCl ₂, 100mM 2-amino-2-methyl-1, ammediol, in the damping fluid of pH 8.7 with

Green I (1: 10,000) or LightCycler Green (10 μ M) mix.On high resolution device, obtain melting curve with 0.1 ℃/second speed.

As mentioned above, with

Green I difference, LightCycler Green can differentiate heteroduplex molecule with the concentration compatible with PCR in melting curve changes.Why illustrated among Fig. 7

GreenI can not differentiate a low reason of unwinding and changing easily.Under the situation that the dna fragmentation that several stability increase progressively exists, compare with LightCycler Green, adopt

The low temperature peak of Green I is very little.An explanation is in separating chain process,

Green I may be released from the low temperature duplex molecule, only combines with the duplex molecule that unwinds under higher temperature.This causes that each follow-up peak is higher than previous, and is also very little even if the peak of the minimum temperature that makes can be observed.The LightCycler Green that exists with much higher saturated level in addition the low temperature duplex molecule have the visible peak.Though LCGreen is when existing near saturated level in the present embodiment, surprisingly, when being diluted to the saturated level of 5-20%, LC Green can detect the low temperature peak.For example, the represented data of Figure 13 are to obtain by the LC Green that uses 1 μ M concentration.Thereby though also do not understand its mechanism, LC Green and multiple other saturable dyes of the present invention are not redistribution in separating chain process.

If the area at each peak and divided by the dose known amounts of every kind of DNA among mensuration Fig. 7 can be estimated the relative sensitivity (Fig. 8) for every kind of DNA.As shown in Figure 8, adopt LightCycler Green to help the low temperature peak that unwinds, and adopt

During Green I, at high temperature observe the remarkable enhancing of signal.

Embodiment 8

In the titration curve and PCR of general double-stranded DNA dyestuff

The mensuration in LightCycler Green usable concentration interval

The general double-stranded DNA dyestuff of 100 nanogram low-molecular-weight dna marks and different concns contains 3mM MgCl final volume 10 microlitres ₂, 50mM Tris, mix in the solution of each 200 μ M of pH 8.3,250 mcg/ml BSA and every kind of dNTP.Sample transfer arrives

Pipe is also measured 40 ℃ fluorescence on the real-time fluorescence meter.With the maximum fluorescence value that every kind of particular dye obtained fluorescence is carried out standardization.

The HTR2A amplicon that uses 152bp contains 3mM Mg final volume 10 microlitres ²⁺50mMTris-HCl, pH 8.3,500 mcg/ml BSA, every kind of each 200 μ M of dNTP, every kind of each 0.5 μ M of primer, 50 nanogram genomic dnas, 0.4 in the solution of the Taq of unit polysaccharase and 88 nanogram TaqStart antibody, be positioned at 2 μ M with extent of dilution and dilute research to the LC Green within the 100 μ M intervals.After 95 ℃ of initial sex change of 10 seconds, with 95 ℃ 0 second, 62 ℃ 2 seconds and 72 ℃ circulation in 20 seconds 40 times.

Go up after the additional temperature condition effect (95 ℃ 0 second, 55 ℃ 0 second), sample unwinds on high resolution device with 0.3 ℃/second slope.

It is compatible with PCR that Fig. 9 A-B has represented The concentration of Green I and LC Green.Under the concentration compatible with PCR,

Green I makes the DNA amount of common existence in PCR latter stage saturated far away.On the contrary, LightCycler Green can use comprising on the interval of concentration widely that makes it saturated.The typical melting curve on 50 times the interval of differing that has shown LightCycler Green concentration among Figure 10.

Embodiment 9

The fluorescence spectrum of Green I and LightCycler Green

With the DNA bonded

Excite and the emmission spectrum of Green I and LightCycler Green are gone up measurement at the photofluorometer (FL-1) of Photon Technology company.3mM MgCl at final volume 60 microlitres that contain 100 nanogram DNA (low-molecular-weight dna mark) ₂, 50mM Tris, add in the solution of each 200 μ M of pH 8.3,250 mcg/ml BSA and every kind of dNTP LightCycler Green (10 μ M) or

Green I (1: 10,000).

Optics with

Green I excites and launches matched well (Figure 11).Even if LightCycler Green with

The optics coupling is bad,

The observed fluorescent signal of last LightCycler Green with the compatible concentration of some PCR than usually from

The observed signal of Green I stronger (data are unexposed).

Embodiment 10

Use the gene type of the betaglobulin of X-axis adjustment and fluorescence variance analysis

Fragment from the regional 110bp that increases of people's betaglobulin of No. 11 karyomit(e)s (accession number NG_000007).This 110bp fragment has comprised common betaglobulin sudden change HbS and the site of HbC.DNA extracts from the dried blood cake of every kind of common genotypic 4 Different Individual.These genotype have comprised 3 homozygotes (AA, SS and CC) and 3 heterozygotes (AS, AC and SC) type.Forward primer and reverse primer are respectively ACACAACTGTGTTCACTAGC (SEQ ID NO:3) and CAACTTCATCCACGTTCACC (SEQ ID NO:4).Per 10 microlitre reaction systems have comprised 50 nanogram genomic dnas, each every kind of primer of 0.5 μ M, 10 μ M LC Green, 3mM MgCl ₂, 50mMTris, pH 8.3,500 mcg/ml BSA, every kind of each 0.2mM of dNTP, 0.04 unit/microlitre Klentaq ^TM(AB Peptides, St.Louis, MO), 88 nanogram TaqStart ^TMAntibody (CloneTech, Palo Alto, CA).The PCR reaction conditions is as follows: preceding 95 ℃ of sex change in 5 seconds circulate; 94 ℃ 0 second, 50 ℃ 2 seconds, 72 ℃ 2 seconds the circulation 35 times, slope is 2 ℃ of per seconds.Each sample is obtained single fluorescent value after extending 2 seconds.After the pcr amplification, sample cools off with-20 ℃ setting speed.And then fast after the cooling, separate on the chain apparatus speed in 16 bit high resolving power of customization and unwind, obtain the fluorescence reading simultaneously continuously from 70 ℃ to 93 ℃ with 0.30 ℃/second.

Along with sample temperature improves, obtain high resolving power melting curve data by measuring fluorescence.Shown the raw data that obtains from 6 kinds of genotypic quadruplicate samples of betaglobulin among Figure 12 A.Note since between the different samples that sample volume difference and the change of kapillary optics cause the amount of fluorescent value change.

Between the sample magnitude difference can by use before the main transformation and afterwards the linear baseline of every curve carry out stdn.Specifically, select two linearity region, one of them is before main the transformation, and one after this.These zones are two straight lines of every curve definitions, top 100% fluorescence straight line and following 0% fluorescence straight line.Fluorescence percentage ratio in the transition process under (between these two zones) each temperature is with calculating apart from percentage ratio between the top and following straight line of inferring.Shown betaglobulin standardization of data result among Figure 12 B.Every kind of genotypic quadruplicate sample obviously condenses together, and is apparent that most near 84-85 ℃.It is accessory deviation that every kind of genotype inside still has some (to note having an appointment 0.2 ℃ discrete range near the 10-20% fluorescent value between the inner quadruplicate sample of genotype) for operation intermediary temperature deviation.This sample deviation can be between two different samples or even take place between twice of same sample different operation.Different preparations comprise the preparation of different salt concn also can causing temperature deviation.Yet for initial at least being similar to, these differences are the shape of influence curve not.

Temperature deviation between the operation can be proofreaied and correct by the temperature axis that moves every curve, and they are overlapping between a given phosphor region like this.For example, select a sample, and will some a quadratic equation be carried out match between phosphor region as standard substance.To every remaining curve, calculate for each interior between described phosphor region point is transformed into needed temperature movement value on the described quadratic equation.Every curve makes described curve overlapping between selected phosphor region with average movement value translation then.The low temperature that heterozygote amplification produces heteroduplex molecule unwinds and changes and the transformation of unwinding of the high temperature of homoduplex molecule.As seeing among Figure 12 C, if moving curve so that its high temperature homoduplex molecular domains (low fluorescence percentage ratio) is overlapping, heteroduplex molecule can be differentiated in the early stage decline under the low temperature more by it.Yet because the curve shape of different homoduplex molecules does not have wide variation, the temperature of different homoduplex molecules moves any difference that may cover between them.

At last, by the fluorescence difference mapping between standardized (and optional ground temperature move) melting curve is easy to observe different genotype most.Select a kind of standard gene type (for example, using betaglobulin wild-type AA) earlier.Shown in Figure 12 D, the difference between every curve and the standard is mapped to temperature subsequently.The standard gene type is 0 (having deducted itself) under all temperature.Other genotype are followed unique route and can be differentiated by the visual pattern coupling.The automatic mode of feature extraction also can be used for determining genotype.In addition, though illustrative embodiment has used saturable dye and heteroduplex molecule to detect, need understand that temperature moves with temperature contrast mapping can be used for not existing gene type under the heteroduplex molecule situation, for example can being used for wherein, genome is haploid gene typing.The example of such hrr gene somatotype comprises hepatitis C gene type, subtype of human papilomavirus gene, HIV gene type and the Bacteria Identification that increases by rDNA.

Can design the single parameter that is associated with genotype.For example, can use standardized curve to determine that different genotype is such as 10% (90% fluorescent value) temperature of unwinding.Remarkable like this some genotype of having distinguished, but can not significantly distinguish other genotype (Figure 12 B).In addition, the maximum slope that can use curve is with differentiation homozygote and heterozygote, but different homozygotes often is similar under maximum slope.At last, can use different areas under a curve (Figure 12 D), but follow different routes but such curve can have similar area with the definition genotype.Though the combination of parameter may confirm to determine it is effectively for the automatization gene type, this technology is very suitable for the visual pattern coupling.

Need understand that other standardisation technique is utilizable and comprises within the scope of the present invention.For example, HR-1 (Idaho Technology, Salt Lake City UT) have the setting that can adjust fluorescent value under pre-determined temperature automatically (for example 40 ℃ the time be 100 fluorescent value), and the melting curve of all samples can be arranged according to identical fluorescent value alignment.When the difference between the stdn of above-mentioned stdn and the control of this machine is to use the stdn of machine control, changes preceding and change the back slope of a curve and do not evened up.

Embodiment 11

The analysis of bigger amplicon

Though short amplicon causes bigger gene type difference usually, LightCycler Green also can be used for the gene type of bigger amplicon.The DNA about 50-500bp of structural domain normal length that unwinds, bigger amplicon, for example the amplicon of 500-800bp has a plurality of structural domains that unwind.Sequence in structural domain changes may not influence unwinding of other structural domains, and the inner observed variation of structural domain can be independent of amplicon length.Thereby, though example in the 400-650bp interval is provided, can there be the upper limit for the size that can be used to scan the PCR product that has the sequence change.

And, because a structural domain unwind it seems be independent of other structural domains in succession, fixed structural domain can be as internal reference to adjust X-axis (temperature axis) because the variation of device or sample operation.Since the difference of melting curve shape, heterozygote each other and and homozygote between can distinguish.The shape of melting curve is by the stability of heteroduplex molecule that exists and homoduplex molecule and/or the kinetics speed definition that unwinds.Owing to there are a plurality of structural domains that unwind in bigger amplicon, change in shape can occur in any part of curve.Locate so that the fixed part of curve is overlapping by the X-axis of adjusting many curves, be easier to distinguish the changing unit of curve.In addition, by the changing unit of superimposed curves, if there is the several genes type, the rest part of curve can be different.The X-axis adjustment in addition can by before the PCR or before unwinding outside each sample adds (1) with reference to nucleic acid, or (2) have the dyestuff of second emission wavelength, and this dyestuff is not with nucleic acid interaction but its fluorescence depends on temperature (as the dyestuff with good temperature factor of Cy5) carries out.Temperature axis moves the position that should be subsequently changes according to unwinding of described contrast nucleic acid or carries out according to the intensity curve of described contrast dye.

Figure 13 A and 14 is for the example of the bigger amplicon of two analyses.Figure 13 A has shown the segmental amplification of people's 5-hydroxytryptamine receptor 2A (HTR2A) gene extron 2 (accession number NM_000621.1) 544bp.Forward primer and reverse primer are respectively CCAGCTCCGGGAGA (SEQ ID NO:5) and CATACAGGATGGTTAACATGG (SEQ ID NO:6).Per 10 microlitre reaction systems comprise 50 nanogram genomic dnas, each 0.5 μ M of every kind of primer, 1 μ M LC Green, 2mM MgCl ₂, 50mM Tris, pH 8.3,500 mcg/ml BSA, every kind of each 0.2mM of dNTP, 0.4 unit K lentaq ^TM(ABPeptides, St.Louis, MO) and 88 nanogram TaqStart ^TMAntibody (CloneTech, Palo Alto, CA).

The PCR reaction conditions is as follows: 92 ℃ 0 second, 60 ℃ 2 seconds, 74 ℃ 25 seconds the circulation 40 times.After the pcr amplification, sample cools off with-20 ℃/second setting speed.Follow hard on quick cooling, separate on the chain apparatus speed in 16 bit high resolving power of customization and unwind, obtain fluorescence simultaneously continuously from 70 ℃ to 93 ℃ with 0.30 ℃/second.

As shown in FIG. 13A, increase and analyze the duplicate sample of every kind of genotype (CC, TC and Tr).Except curve overlapping between 10 and 20%, the standardization of data and temperature moved as described in embodiment 10 carry out.Figure 13 B represents the prediction of homoduplex molecule unwind figure and the pleomorphism site in lesser temps unwinds structural domain.Experimental data has shown two apparent structural domains that unwind.All genotype are similar in comparatively high temps unwinds structural domain.Genotype there are differences in lesser temps unwinds structural domain, wherein heterozygote shows the unwind typical behavior of heteroduplex molecule of low temperature, show as heterozygote curve and the low temperature homozygote curved intersection of unwinding, and approach the homozygote of higher temperature along with temperature raises.

Figure 14 represents the difference curve of cystic fibrosis spanning transduction membrane regulator (CFTR) gene extron 10 (accession number M55115) 612bp fragment amplification.Forward primer and reverse primer are respectively AGAATATACACTTCTGCTTAG (SEQ ID NO:7) and TATCACTATATGCATGC (SEQ ID NO:8).Per 10 microlitre reaction systems comprise 50 nanogram genomic dnas, each 0.5 μ M of every kind of primer, 10 μ M LC Green, 3mM MgCl ₂, 50mM Tris, pH 8.3,500 mcg/ml BSA, every kind of each 0.2mM of dNTP, 0.4 unit K lentaq ^TM(AB Peptides, St.Louis, MO) and 88 nanogram TaqStart ^TMAntibody (CloneTech, Palo Alto, CA).The PCR reaction conditions is as follows: 89 ℃ 0 second, 58 ℃ 8 seconds, 74 ℃ 35 seconds the circulation 35 times.Each sample is obtained single fluorescent value after 35 seconds extend.After the pcr amplification, sample cools off with-20 ℃/second setting speed.Follow hard on quick cooling, separate on the chain apparatus speed in 16 bit high resolving power of customization and unwind, obtain fluorescence simultaneously continuously from 60 ℃ to 87 ℃ with 0.30 ℃/second.In this example, when the middle portion (30-40% fluorescence) of curve was used for the X-axis adjustment, it is best that heterozygote is distinguished effect.At last, the fluorescence that deducts every curve of from the wild-type curve has obtained disparity map as shown in figure 14.Every kind of sequence changes obvious different and can distinguish all genotype with wild-type.

Embodiment 12

Adopt target detect and the frequency multiplexing technique of LightCycler Green

LightCycler Green can be as donor to excite the acceptor dye that links to each other with oligonucleotide probe.Because LightCycler Green can be to reach or to be used for high-density in conjunction with hybridization probe (about 2 dye molecules of per 3 base pairs) near saturated concentration, the dyestuff that runs through the double-stranded DNA total length can be used for FRET (fluorescence resonance energy transfer).In reaction system, adding probe before the PCR with acceptor dye, amplification and with the situation of product hybridization under detect.LightCycler Green has promoted that with high-density and combining of duplex molecule this has produced high-intensity acceptor fluorescence to the exciting of acceptor dye on the probe.In the past, used dyestuff also only to produce low-level acceptor fluorescence with high bp/ dyestuff ratio.

Adopt multiple probe can carry out the polychrome experiment.For example, whole amplicon unwinds and can monitor in 470 nanometers, the emission light of fluorescein-labelled probe can be monitored in 515 nanometers, HEX label probe (with the different dna fragmentation hybridization in the primer) is the 3rd wavelength place monitoring, and TET label probe (with another different dna fragmentations hybridization in the primer) is the 4th wavelength place monitoring.As known in this area, color compensation is in order to deconvolute to four kinds of signals of eclipsed.The result is that first kind of signal can be used for the sudden change scanning to whole amplicon, and second, third makes the gene type in the inner smaller area of amplicon territory become possibility with the 4th kind of signal.

Embodiment 13

Other double-stranded DNA combination dyes

Table 1 has been summed up the character and the feature of 37 kinds of different dyes.Under the situation that heterozygote Δ F508 genotype is increased, the dyestuff of 12 kinds of detections does not produce heteroduplex molecule peak (invalid).Use 25 kinds in 37 kinds of different dyes to detect heteroduplex molecule peak (effectively).When using LightCycler Green, produced the strongest heteroduplex molecule signal, use other several dyestuffs also to show good heteroduplex molecule signal.Most of dyestuff that shows heteroduplex molecule is saturated or compatible with PCR during near saturation concentration.This dependency allows carrying out reasonable prediction by the dyestuff that curve analysis detects heteroduplex molecule.50% saturation ratio provides the reasonable prediction to heteroduplex molecule.Though missed some effective dyestuffs, about 80%, 90% or even 99% saturation ratio per-cent can be used for differentiating the dyestuff that can detect heteroduplex molecule.

Simultaneously, many effective asymmetric cyanine dyess have low bp/ dyestuff ratio when 50% saturation ratio, particularly are lower than the 4.0bp/ dyestuff and more specifically are lower than the 2.0bp/ dyestuff.Think that at first because this low bp/ dyestuff ratio has prevented or minimized redistribution at the commitment that unwinds, and heteroduplex molecule is easier to detect like this.Yet, as shown in table 1, even if prosper some dyestuff has sufficiently high bp/ dyestuff ratio, still can detect heteroduplex molecule.And, under the concentration that fully is lower than saturated level, still can detect heteroduplex molecule even if having the dyestuff of low bp/ dyestuff ratio.Thereby low bp/ dyestuff ratio is a factor when discriminating forms suitable dyestuff for heteroduplex molecule.

Preferably, when molar extinction coefficient is high (＞30,000), fluorescence is stronger.Best two kinds of dyestuffs (in the heteroduplex molecule context of detection) show at the maximum excitation wavelength of 430-455 nanometers and the maximum emission wavelength of 450-480 nanometers.Usually, these are than the shorter wavelength of normally used wavelength in the fluorescein passage of typical real-time clock.Nonetheless, the fluorescent signal ratio from LightCycler Green derives from

The signal of Green I is stronger, considers

Green I has and much better Wavelength matched of the spectral filter that uses, and this discovery is surprising (Figure 11).

Embodiment 14

Preparation and use based on the dna binding dye of pyrimidyl

Some embodiment that has prepared dyestuff with following pyrimidyl core texture:

Wherein

X, R ², R ³, and R ⁵By the definition of here structural formula I being done, the definition of being done among B such as the structural formula V.

Though exist multiple preparation to have the method for the dyestuff of this structural formula, a kind of method is as follows:

Wherein compound 6 has commercial stock, maybe can prepare by ordinary method.Compound 7a comprises lithium alkylide, aromatic series and fatty amine, K by under neutrality or alkaline condition ₂CO ₃Deng, 6 alkylations are prepared to compound on N (3) position to use alkylating reagent as alkyl halide, alkyl-sulphate etc.Similarly, compound 7a is by being prepared compound 6 arylations on N (3) position by the aromatic series linked reaction as the aromatic halide of metallic compounds such as copper, palladium, platinum and other catalysts, borate etc.Compound 7b is by such as using hydrogen peroxide, comprising that the conventional oxidizing reaction of the peroxy acid etc. of m-CPBA is prepared from compound 7a.In some cases, compound 7a and compound 7b have commercial stock.Compound 8 has commercial stock, or is prepared by ordinary methods such as locational alkylation of N (1) or arylation as described herein.In addition, compound 8 is by 1 of suitable replacement, and the polymerization of 3-diketone and urea or thiocarbamide is prepared.Further, on C (2) position, have as herein defined and comprise that the compound 8 of leavings groups such as halogenide, alkyl sulphonyl, aryl sulfonyl can be by introducing in C (2) position as modifying based on the nucleophilic reagent of the nucleophilic reagent of nitrogen, oxygen and sulphur under neutrality or alkaline condition.Further, have in C (2) position oxygen or sulphur nucleophilic group compound 8 can by under neutrality or the alkaline condition on C (2) hydroxyl or sulfydryl introducing alkide or acylate modify.Compound 9 reacts under neutrality or alkaline condition by compound 7 and compound 8 as described herein and is prepared.

Exemplary compounds such as described here being prepared with described structural formula adopt triethylamine-ammonium acetate to carry out purifying as moving phase by HPLC, separate with the form of its corresponding acetate.These exemplary compounds are illustrated in table 2.

Table 2

Compound D 6 is passed through at first 4-methylpyrimidine and (3-bromopropyl) TMA (TriMethylAmine) bromine reaction under the acetonitrile reflux conditions; Back flow reaction is prepared in argon gas existing under anhydrous pyridine and the triethylamine condition for product in the acetonitrile of gained (compd A 6) and 3-methyl-2-methylsulfonyl benzothiazole iodide (Aldrich).

Compd E 6 is prepared according to the general step that is used for from 3-methyl-2-methylsulfonyl benzoxazole anchor iodide (being prepared by 2-methylsulfonyl benzothiazole and dimethyl sulfate reactant salt) and compd A 6 preparation Compound D 6.

Compound G5 is prepared according to the general step that is used for from 2-methylthio group-3-phenyl benzothiazole iodide (Aldrich) and compd A 6 preparation Compound D 6.

Compound H 5 is prepared according to the general step that is used for from 5-difluoro methylsulfonyl-3-methyl-2-first sulphur benzothiazole methylsulfuric acid ester (being prepared by 5-difluoro methylsulfonyl-2-first sulphur benzothiazole (Aldrich) and dimethyl sulfate reactant salt) and compd A 6 preparation Compound D 6.

Compound P 6 is prepared according to the general step that is used for from 5-chloro-2-(methylthio group)-3-(3-sulfopropyl)-benzothiazole oxyhydroxide (Aldrich) and compd A 6 preparation Compound D 6.

Compound R 6 is prepared according to the general step that is used for from 6-amino-3-methyl-2-first sulphur benzothiazole methylsulfuric acid ester (being prepared by 6-amino-2-first sulphur benzothiazole (Aldrich) and dimethyl sulfate reactant salt) and compd A 6 preparation Compound D 6.

Compound Y6 is according to being used for from 3-methyl-2-methylsulfonyl naphtho-[1; 2-d] oxazole methylsulfuric acid ester is (by 2-methylsulfonyl naphtho-[1; 2-d] oxazole (Chem Bridge Product List; San Diego CA) is prepared with the dimethyl sulfate reactant salt) and compd A 6 general step for preparing Compound D 6 be prepared.

Compound Z6 is according to being used for from 3-methyl-2-methylsulfonyl naphtho-[1; 2-d] the thiazole methyl sulfuric ester is (by 2-methylsulfonyl naphtho-[1; 2-d] thiazole (Specs, Rijswijk, The Netherlands) and dimethyl sulfate reactant salt be prepared) and compd A 6 general step for preparing Compound D 6 be prepared.

Compound D 8 is by reflux N-phenylthiourea and 2 in HCl/ ethanol, and 4-diacetylmethane solution is prepared.The backflow reaction overnight is prepared with generation Compound D 8 in chloroform/methanol (10: 1) existing under the condition of triethylamine for the pyrimidine thioketone that obtains and 3-methyl-2-methylsulfonyl benzothiazole iodide.

Compound I 5, K5, L5, G8, K8, L8,18, M8, N8, C8, E8, F7 and O8 can be prepared by above-mentioned similar methods.These dyestuffs estimate can be used to detect heteroduplex molecule.

Cyanine dyes based on pyrimidine described here, for example G5, H5, D6, E6, P6, R6, Y6, Z6 and D8 are reactive monoazo dyestuffs and the detection that can be used for heteroduplex molecule, sudden change scanner uni gene type.Table 3 has been summed up the result who uses these dyestuffs in heteroduplex molecule detects.It will be noted that for LC Green heteroduplex molecule per-cent is than high in the table 1 in the table 3.This difference may be owing to used bigger amplicon during data in the acquisition table 3.

Table 3

Dyestuff	Ex/Em 1	The compatible %Sat of maximum PCR 2	％Het 3
				LC Green	450/469	＞99％	20.5％
PO-PRO TM-1	438/457	100％	19.6％
				BO-PRO TM-1	438/457	100％	17.1％
D6	457/471	92％	23.3％
				E6	425/454	＞99％	15.0％
P6	464/490	100％	21.0％
				R6	453/470	＞90％	15.0％
G5	442-458/475	100％	20.0％
				H5	444/459	100％	22.5％
Y6	439/477-515	100％	21.0％
				Z6	469/494-526	100％	13.4％
D8	453-457/471	100％	19.8％

1. (3mM MgCl in the PCR damping fluid ₂50mMTris, pH 8.3, each 200 μ M of every kind of dNTP, 500 mcg/ml BSA) the maximum excitation wavelength (Ex) and the maximum emission wavelength (Em) that use the dyestuff of 2.5 μ M bp (100 nanograms/60 microlitres) double-stranded DNA and the compatible concentration of maximum PCR on photofluorometer, to obtain.Some dyestuff is because emission or excitation wavelength peak broad have interval of wavelength.

2. all under the condition that has 15 μ M bp DNA (100 nanogram double-stranded DNAs/10 microlitres) and PCR damping fluid, can be present in the PCR mixed solution peak concentration that amplification is not produced the dyestuff that obviously suppresses, represent with the fluorescence per-cent of comparing with the fluorescence of the identical dyestuff of saturation concentration (concentration of possible maximum fluorescence intensity promptly is provided).

3. use the del F508 heterozygote melting curve that obtains with 0.3 ℃ of/second heating slope, excite the peak area per-cent of the heteroduplex molecule characteristic peak of measuring with 450-530 nanometer detection optics with the 420-490 nanometer.The amplicon length of using in the experiment of this group is 57bp, produces by primer GGCACCATTAAAGAAAATAT (SEQ ID NO:23) and TCTGTATCTATATTCATCATAGG (SEQ ID NO:24).Write down largest percentage.

Embodiment 15

Be used for genotype high resolving power curve analysis relatively

Dyestuff of the present invention can be used for determining whether any two individualities have identical allelotrope on a gene fragment.In the former example, a genotype with reference to sample (comprising definite allelotrope, heterozygosity and haplotype) is known.The high resolving power curve analysis in some applications, do not need to know definite genotype, as long as can determine whether (or unknown originate) sample of another individuality is identical with reference with reference to sample.An illustrative example is the allelic discriminating of HLA total among the family member.

Human leucocyte antigen (HLA) (HLA) is the cell surface protein of leukocyte cell and its hetero-organization of body, plays a crucial role at immunity identification and transplantation tolerance or in repelling.For organ transplantation, the HLA allelotrope pairing between donor and the acceptor is important.HLA albumen forms two primary categories: I class and II class.Each classification has a plurality of genes encodings.Being used for of generally acknowledging at present determine the technology of the HLA allelotype of tissue comprise serotype with specific antibody reagent, with the hybridization of nucleic acid probe and the direct order-checking of HLA gene.Because need to detect a large amount of genes and site, the cost of measuring the HLA allelotype has surpassed everyone 1,000 dollar.When donor and acceptor were irrelevant, the complete gene type of HLA was essential.Yet, the chance that the 25%HLA that has an appointment between the compatriot mates fully, so when the HLA coupling illustrates that it is feasible, the organ transplantation between the preferred compatriot.In this case, only need the total identical HLA allelotrope of proof donor and acceptor relatives.There is no need to measure described total allelic stric consistency.

The genome DNA sample of CEPH/ family Utah family 1331 derives from Coriell institute.3 generations 17 people are arranged in this family, comprise 4 grand parents, two father and mother and 11 children (Figure 15 has shown the pedigree chart of family 1331).Have the homozygote genotype HLA-ABM15 (0101) that knows and two other samples of BM16 (0202) and also derive from Coriell.

The amplification of two exons of HLA-A is following to be carried out: I class HLA gene is so similar on the total length of its coding exon, so that is difficult to design the PCR primer of the I genoid that only increases the HLA-A gene and do not increase relevant.Adopted the nest-type PRC strategy, wherein a big fragment (948bp) of first round PCR specific amplification HLA-A gene is carried out the amplification second time with inboard primer to product subsequently.Be used for the primer of first round PCR and HLA-A introne 1 (forward primer 5 '-GAAAC (C/G) GCCTCTG (C/T) GGGGAGAAGCAA (SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12)) and intron 4 (reverse primer 5 '-TGTTGGTCCCAATTGTCTCCCCTC (SEQ ID NO:13)) hybridization.Second takes turns and has used forward primer 5 ' AGCCGCGCC (G/T) GGAAGAGGGTCG (SEQ ID NO:14, SEQ ID NO:15) and reverse primer 5 ' GGCCGGGGTCACTCACCG (SEQ ID NO:16) the 335bp fragment with amplification HLA-A exon 2 among the PCR.Forward primer 5 ' CCC (G/A) GGTTGGTCGGGGC (SEQ ID NO:17, SEQ IDNO:18) and reverse primer 5 ' ATCAG (G/T) GAGGCGCCCCGTG (SEQ ID NO:19, SEQ ID NO:20) be used to increase 366bp fragment of HLA-A exon 3.In the primer sequence of present embodiment, the described primer of (N/N ') representative is the mixture that has the nucleotide sequence of the N of equal percentage and N ' in this position.For example, as sequence No 17 and No 18 representatives, the segmental forward primer of the 335bp of HLA-A exon 2 contains the equal blend thing of two kinds of Nucleotide (G or A) on the 4th position.The forward primer of HLA-A introne 1 has 2 such sites, thereby is the equal blend thing as 4 kinds of Nucleotide of sequence 9,10,11,12 representatives.

All PCR use Roche

In glass capillary, carry out.First round PCR contains 0.5 μ M forward and reverse primer, contains damping fluid (the 3mM Mg of 50 nanogram genomic dnas in 10 microlitre systems ⁺⁺, the D6 dyestuff of 50mMTris-HCl pH 8.3,500 mcg/ml BSA and 20-).Cycling condition be 94 ℃ 20 seconds, subsequently 94 ℃ 1 second, 62 ℃ 0 second, 72 ℃ 1 minute the circulation 40 times.Second takes turns nest-type PRC comprises 0.25 μ M forward and reverse primer, is containing 2mM Mg ⁺⁺Same buffer in 1/10000 first round PCR product.Cycling condition be 94 ℃ 5 seconds, subsequently 94 ℃ 1 second, 65 ℃ 0 second, 72 ℃ 8 seconds the circulation 25 times.

After the amplification, glass capillary is transferred to high resolving power and is separated on the chain apparatus HR-1, and unwinds for the second time.Sample is heated to 95 ℃ and per 40 seconds with 0.3 ℃/second speed from 60 ℃ and obtains fluorescence (450 nanometer excitation wavelengths/470 nanometer emission wavelengths) and measured temperature (Figure 16 A-B).Nido order-checking product checks order with ABI3700.4.0 the Sequencher of version is used for sequential analysis.

The following mensuration of the degree of agreement of curve analysis and sequencing result: be clustered into 6 different groups (Figure 16 A-B) from the exon 2 of 17 members amplification of CEPH/ family Utah family 1331 and the curve analysis of exon 3 PCR product.This explanation has 6 kinds of different genotype in this family.Exon 2 and exon 3 PCR product are checked order, and the result confirmed curve analysis that 6 kinds of genotype that identify are: family member 1,4,7,12 HLA-A 02011/3101 (being called genotype AB here); Family member 3,5,6,11,17 HLA-A 3101/2402101 (genotype BC); Family member 2,9,10,16 HLA-A02011/2402101 (genotype AC); Family member 13,14 HLA-A02011/03011 (genotype AD); Family member 8 HLA-A 02011/02011 (frequency of genotypes AA) and family member's 15 HLA-A2402101/01011 (genotype CE) (Figure 16 A-B has shown the result of exon 2).

In some cases,, may show consistent or approaching consistent melting curve though have different genotype from compatriot's amplified production.In this case, before first round PCR, mix genomic dna, carry out two amplification step subsequently, and curve analysis can be distinguished identical with different genotype from two compatriot.Under special circumstances, if the compatriot has an identical genotype, the blended melting curve can with those carry out separately identical.If the compatriot has different genotype, the blended melting curve can be different with independent melting curve so.Combined experiments in every group confirms the total identical genotype of every group member.

Another example of hybrid analysis technique has been described with two homozygote sample BM15 (0101) and BM16 (0201).In this case, described two allelotrope are studded with 15 nucleotide differences altogether on the total length of HLA-A exon 2, but both show similar melting curve.Because 15 mispairing of existence on the allos hybrid molecule that is produced by HLA-A exon 2 biased sample PCR, the melting curve of biased sample obviously move to left (lower melting temperature(Tm)) (seeing Figure 17).

Embodiment 16

Use the amplification of saturable dye to monitor in real time

With the 60bp fragment of dividing other forward and reverse primer ACCAGGCTCTACAGTAA (SEQ ID NO:21) and GTTAAATGCATCAGAAG (SEQ ID NO 22) to increase the HTR2A gene.Except the change to loop parameter (is used

95 ℃ 0 second, 62 ℃ 2 seconds, 74 ℃ 20 seconds) outside, use the reagent described in the embodiment 11 to increase.There is different concns in the reaction mixture independently of one another Green I, With

16.Each amplification cycles obtains fluorescence data, until circulation 40 times.Obtained the fluorescence point of crossing (Cp) of following secondary derivative maximum value calculation with amplification figure (draw on the X-axis on the relative Y-axis of cycle number and draw fluorescence intensity).

Table 4

When the inhibitor that exists in the reaction influenced amplification efficiency, the Cp value expectation of representing signal wherein to exceed the cycle number of background can improve.Yet under these experimental conditions, what the inhibition of the dyestuff of the amount of being incremented caused is not the progressively raising of Cp value, but the amplification stop fully suddenly.Because big or small less (having caused comparing littler signal) of amplicon with bigger amplicon, Green I dyestuff can only be used for real-time monitoring in the double strength interval.On the contrary, With 16 can use in 8 times of concentration intervals.Estimating that many saturable dyes have can be used to increase the wide concentration interval of monitoring in real time.

Above-mentioned discussion discloses and described only is example row embodiment of the present invention.Be proficient in those skilled in the art and easily recognize, can carry out various changes, improvement and change and do not leave it as the defined the spirit and scope of the present invention of following claims from such discussion and the accompanying drawing of following and claim.

Here all that quoted are with reference to by with reference to intactly in conjunction with in this manual.

Sequence table

＜110〉G.T. Witter fertile (Wittwer, Carl T.)

G. Reed (Reed, Gudrun)

V.E. Du Qiaole (Dujols, Virginie E.)

L. all (Zhou, Luming)

＜120〉the amplicon melting analysis of use saturable dye

<130>7475-73421

<140>PCT/US2003/033429

<141>2003-10-22

<150>US 60/439,978

<151>2003-01-14

<150>US 60/420,717

<151>2002-10-23

<160>24

<170>PatentIn version 3.2

<210>1

<211>20

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>1

ggcaccatta aagaaaatat 20

<210>2

<211>18

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>2

tcatcatagg aaacacca 18

<210>3

<211>20

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>3

acacaactgt gttcactagc 20

<210>4

<211>20

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>4

caacttcatc cacgttcacc 20

<210>5

<211>14

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>5

ccagctccgg gaga 14

<210>6

<211>21

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>6

catacaggat ggttaacatg g 21

<210>7

<211>21

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>7

agaatataca cttctgctta g 21

<210>8

<211>17

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>8

tatcactata tgcatgc 17

<210>9

<211>26

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>9

gaaaccgcct ctgcggggag aagcaa 26

<210>10

<211>26

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>10

gaaacggcct ctgcggggag aagcaa 26

<210>11

<211>26

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>11

gaaaccgcct ctgtggggag aagcaa 26

<210>12

<211>26

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>12

gaaacggcct ctgtggggag aagcaa 26

<210>13

<211>24

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>13

tgttggtccc aattgtctcc cctc 24

<210>14

<211>22

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>14

agccgcgccg ggaagagggt cg 22

<210>15

<211>22

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>15

agccgcgcct ggaagagggt cg 22

<210>16

<211>18

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>16

ggccggggtc actcaccg 18

<210>17

<211>17

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>17

cccgggttgg tcggggc 17

<210>18

<211>17

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>18

cccaggttgg tcggggc 17

<210>19

<211>19

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>19

atcagggagg cgccccgtg 19

<210>20

<211>19

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>20

atcagtgagg cgccccgtg 19

<210>21

<211>17

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>21

accaggctct acagtaa 17

<210>22

<211>17

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>22

gttaaatgca tcagaag 17

<210>23

<211>20

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>23

ggcaccatta aagaaaatat 20

<210>24

<211>23

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>24

tctgtatcta tattcatcat agg 23

Claims (12)

1. the method for a pcr analysis is characterized in that, said method comprising the steps of:

The sample mix of the double-stranded DNA combination dye that will under the compatible concentration of maximum PCR, have at least 50% saturated per-cent and the primer that contains target nucleic acid and be provided with for amplifying target nucleic acid,

Amplifying target nucleic acid under the condition that has described double-stranded DNA combination dye, and

Monitor the fluorescence of described double-stranded DNA combination dye, wherein said monitoring step comprises:

The target nucleic acid of amplification is unwind with the generation melting curve, and

Shape sldh gene type by melting curve.

2. the method for claim 1 is characterized in that, described method is further comprising the steps of:

Value to melting curve carries out standardization,

At least one other target nucleic acid is repeated mixing, amplification, curve generation, standardization step, and

Standard of comparison is dissolved the chain curve.

3. method as claimed in claim 1 or 2 is characterized in that, wherein said dyestuff be selected from following material: LCGreen, Gel Star,

16, PO-PRO ^TM-1, JO-PRO ^TM-1, BO-PRO ^TM-1,

44,

45, POPO ^TM-3, 12, TOTO ^TM-3,

Blue,

-3, 43,

11,

13,

15, BOBO ^TM-3, LO-PRO ^TM-1,

23, TO-

-1,

20, BOBO ^TM-1, POPO ^TM-1, G5, H5, D6, E6, P6, R6, Y6, Z6 and D8.

4. the method for claim 1, it is characterized in that described monitoring step is with the photofluorometer between the emission wavelength detection zone with the excitation wavelength interval of 450-490 nanometer and 510-530 nanometer and has 410-465 nanometer maximum excitation wavelength and the dyestuff of 450-500 nanometer maximum emission wavelength carries out.

5. the method for claim 1 is characterized in that, described method comprises sudden change scanning, and described method also is included on second sample repeating step amplification and monitoring step with acquisition second melting curve, and more described melting curve.

6. the method for claim 1, it is characterized in that, described sample also comprises the probe of being arranged to target nucleic acid hybridization, described probe uses the acceptor dye mark with the FRET (fluorescence resonance energy transfer) of acceptance from the double-stranded DNA combination dye, and comprises the step of monitoring from the fluorescence of described acceptor dye.

7. the method for claim 1 is characterized in that, described monitoring step comprises

Use described melting curve to determine whether described target nucleic acid has the sequence identical with second nucleic acid.

8. the method for a pcr analysis is characterized in that, said method comprising the steps of:

A kind of double-stranded DNA combination dye that has at least 50% saturated per-cent under the compatible concentration of maximum PCR and target nucleic acid and the primer that is provided with for amplifying target nucleic acid are mixed,

Amplifying target nucleic acid under the condition that has described double-stranded DNA combination dye,

Monitor the fluorescence of described double-stranded DNA combination dye,

Generate the melting curve of described target nucleic acid,

Melting curve is carried out standardization,

To another one target nucleic acid at least repeat to mix, amplification, curve generates and the step of standardization, and

The melting curve of standard of comparisonization.

9. method as claimed in claim 8 is characterized in that, described method also comprises the step to the drawing of the fluorescence difference between the standardized curve.

10. method as claimed in claim 8 is characterized in that, described method also comprises by the part of overlapping every curve carries out the step that temperature moves to melting curve.

11. the method for claim 1 is characterized in that, described double-stranded DNA combination dye is a kind of compound with following structural formula

Wherein

The part representative is chosen the condensed monocycle that replaces wantonly or is encircled aromatic nucleus or optional condensed monocycle or the nitrogenous hetero-aromatic ring of polycyclic that replaces more;

X is oxygen, sulphur, selenium, tellurium or is selected from C (CH ₃) ₂And NR ¹Group, R wherein ¹Be hydrogen or C _1-6Alkyl;

R ²Be selected from C _1-6Alkyl, C _3-8Cycloalkyl, aryl, aryl (C _1-2Alkyl), hydroxyalkyl, alkoxyalkyl, aminoalkyl, alkyl monosubstituted amino alkyl and dialkyl aminoalkyl, trialkyl ammonium alkyl, thiazolinyl carboxylate radical, thiazolinyl carboxylic acid amides, olefin sulfonic acid root, alkyl sulfonic ester, the optional heteroatomic aliphatic portion that contains that contains heteroatomic loop section or optional replacement that replaces;

T=0 or 1;

Z is selected from 0 or 1 charge number;

R ³Be selected from hydrogen, C _1-6Alkyl or-C (O) Ph;

R ⁹And R ¹⁰Independently be selected from hydrogen and C separately _1-6Alkyl;

N=0,1 or 2; And

Q is the heterocycle that is selected from following structure:

R wherein ⁴, R ⁵, R ⁶, R ⁷And R ⁸Independently be selected from hydrogen, halogen, alkyl, cycloalkyl, assorted alkyl, Heterocyclylalkyl, thiazolinyl, polyalkenyl, alkynyl, carbene base, alkene alkynyl, aryl, heteroaryl, alkoxyl group, alkylthio or dialkyl amido, wherein every kind can be optionally substituted; Contain heteroatomic aliphatic portion or contain heteroatomic loop section; The bridging dyestuff; And active group, wherein every kind optional contains a quartemary ammonium part.

12. method as claimed in claim 7 is characterized in that, described target nucleic acid is to be homologous genes seat from the HLA gene of second individuality from a locus of the HLA gene of first individuality and described second kind of nucleic acid.

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