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WO1998011433A1 - Methode d'analyse d'un adn - Google Patents

Methode d'analyse d'un adn Download PDF

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Publication number
WO1998011433A1
WO1998011433A1 PCT/AU1997/000595 AU9700595W WO9811433A1 WO 1998011433 A1 WO1998011433 A1 WO 1998011433A1 AU 9700595 W AU9700595 W AU 9700595W WO 9811433 A1 WO9811433 A1 WO 9811433A1
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WO
WIPO (PCT)
Prior art keywords
dna
temperature
temperatures
absorbance
optical density
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PCT/AU1997/000595
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English (en)
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WO1998011433A9 (fr
Inventor
Darryl John Rowe
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Varian Australia Pty. Ltd.
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Filing date
Publication date
Application filed by Varian Australia Pty. Ltd. filed Critical Varian Australia Pty. Ltd.
Priority to AU41052/97A priority Critical patent/AU4105297A/en
Publication of WO1998011433A1 publication Critical patent/WO1998011433A1/fr
Publication of WO1998011433A9 publication Critical patent/WO1998011433A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the present invention relates generally to a method of characterising DNA. More particularly, the present invention contemplates a method of detecting base changes in DNA by determining the optical density at one or more temperatures. Particularly, the method utilises the differential optical densities of single-stranded DNA (ssDNA) and double stranded DNA (dsDNA) preferably to characterise the length and denaturation and renaturation temperatures of melting domains.
  • ssDNA single-stranded DNA
  • dsDNA double stranded DNA
  • DGGE Denaturing gradient gel electrophoresis
  • TGGE thermal gradient gel electrophoresis
  • SSCP- PCR single-stranded conformational polymorphisms
  • Other techniques which are utilised for the detection of DNA mutations include single-stranded conformational polymorphisms (SSCP- PCR) (for example, David et al 1993) [Orita et al, 1989] and chemical cleavage (Fodde and Losekoot 1994).
  • one aspect of the present invention provides a method of characterising DNA including: providing a source of DNA; subjecting the DNA to a pre-determined temperature sufficient to cause a portion of the DNA to denature or to renature; and determining the optical density of the DNA at the pre-determined temperature.
  • DNA melting refers to the thermal or chemical denaturation of dsDNA.
  • T m denaturation temperature
  • the segments of dsDNA that become single-stranded as the denaturation thresholds are exceeded are commonly referred to as melting domains.
  • the DNA may be renatured.
  • the pre-determined temperature for measuring the optical density is a denaturation temperature indicating a melting domain.
  • Spectrophotometric typing of DNA in the present invention may characterise dsDNA with a defined length by determining the denaturation or renaturation temperature and length of melting-domains.
  • a melting-domain within dsDNA denatures as its threshold temperature is exceeded.
  • the denaturation-temperature is governed by complex intermolecular and intramolecular interactions, these interactions are largely determined by the composition and order of nucleotides within the dsDNA.
  • the optical properties of DNA change as it denatures. Single-stranded DNA in aqueous solution absorbs approximately 37% more light in the UV range of the spectrum than dsDNA with the same nucleotide content (Fielder 1982).
  • the optical density increases as denaturation occurs, producing discrete, quantifiable changes in absorbance which are directly proportional to the length of disassociated strands. Conversely, the optical density will decrease as renaturation occurs.
  • This method of the present invention which includes characterising the DNA may include determining base changes in the DNA to determine length of melting domains and/or the denaturation or renaturation temperatures of the individual samples.
  • the DNA samples may be double-stranded DNA and preferably the DNA sample is in an aqueous solution such as water, or phosphate buffered saline (PBS).
  • the source of DNA may derive from any source including a plasmid, bacteriophage such as lambda, T2 or T4, cDNA or genomic DNA.
  • the DNA may be reduced in length by any method including chemical or physical means such as digestion with restriction enzymes or by shearing by physical forces.
  • the DNA may also undergo a purification step such as by electrophoresis or by chromatography so that all DNA tested is substantially the same size.
  • the DNA is less than about 2kb however, any length of DNA can be used.
  • DNA amplified by the polymerase chain reaction may also be a source of DNA. PCR may be conducted in a separate vessel such as microfuge tube or it may be conducted directly within a cuvette used to measure the optical density. The amount of DNA for characterisation using a 300 ⁇ l cuvette may be synthesised in any efficient 50 ⁇ l polymerase chain reaction. The single and unincorporated nucleotides present following PCR amplification of dsDNA may have no effect on the measurement of the changes in optical density.
  • the DNA sample may undergo initial purification and identification to locate the gene of interest before the DNA is subjected to the method of the present invention for further characterisation.
  • the purification step may also yield fragments of the same length or size.
  • the determination of optical density may be a change in the absorbance at a predetermined wavelength.
  • the change in absorbance may be standardised and measured as a ratio of the change in absorbance and a change in temperature over a varying temperature range.
  • the optical density may be a measure of the absorbance and may be measured in a spectrophotometer including a centrifugal spectrophotometer or a conventional spectrophotometer.
  • Other methods of measuring changes in optical density include incorporation of interchelating florescent agents such as ethidium bromide and a commonly used dye from Hoechts could be used to quantify the length of DNA that becomes single stranded as dsDNA denatures. Both agents destabilize the helix to some degree.
  • the wavelength for measuring optical density is in the range of about 250 to 300 nm and most preferably the wavelength is about 260 nm.
  • Centrifugal spectrophotometry may be computerised so as to improve the handling and time efficient characterisation of DNA.
  • the use of a centrifugal spectrophotometer allows for automation of the method and thereby provides for the capacity of analysing large quantities of samples.
  • optical density of the sample is read at varying temperatures.
  • the temperature range may include at least one temperature at which a portion of the DNA sample denatures (denaturing temperature) or renatures and this is generally associated with the sequence of each strand, the intramolecular bond stability and the base stacking between successive turns within the double-stranded helix.
  • the temperature range includes several denaturing temperatures indicating several melting domains.
  • the temperature at which the DNA denatures may indicate at least one melting domain in the DNA where the DNA sample is read at one temperature, only a portion of the DNA may be denatured.
  • the temperature range will include more than one denaturing temperature indicating more than one melting domain.
  • differences in samples may indicate differences in denaturing temperature and lengths of melting domains.
  • the temperature range may be varied in a continuous manner or in a stepwise manner incorporating several ranges. Preferably, the temperature is increased at 1°C/min.
  • the temperature may be applied by any method including the use of a heat block, hot water bath or hot air.
  • a preferred aspect of the present invention further includes increasing the yield of homoduplex molecules following PCR amplification.
  • the present invention provides a method of characterising DNA including: providing a source of DNA by PCR amplification; subjecting the PCR amplification to conditions favouring homoduplex DNA molecule formation; subjecting the DNA to one or more temperatures; and determining optical density of the DNA at each temperature.
  • the PCR-amplification of heterozygous alleles in nuclear DNA produce four different types of molecules. Two molecules with perfect complementarity, these are referred to as homoduplex molecules and two heteroduplex molecules from annealing of the near-complimentary strands amplified from the different alleles, (Sheffield et al 1990) resulting in complicated thermal denaturing profiles.
  • PCR sample may be immediately heated to 100°C for 3 minutes then plunged into ice where it remains for 4 minutes. This rapid cooling favours annealing of primers to their compliment over annealing of the longer PCR-synthesised DNA to its compliment [need reference].
  • Klenow DNA-polymerase or T-7 DNA-polymerase is added and incubated at 37°C for 5 minutes to synthesise the newly primed complimentary strand.
  • Samples that consist of two homoduplexes prepared by this method produce an absorbance versus temperature curve that is the sum of the absorbances of each of the two homoduplex molecules.
  • the melting temperature of polymorphic melting-domains will differ between each molecule. Assuming that the concentration of each homoduplex is slightly different, the relative absorbance of each can be determined from the curve. The relative increase in absorbance with denaturation of analogous polymorphic melting domains, equals the relative absorbance of each molecule. By subtracting the absorbance due to one homoduplex from the absorbance versus temperature curve we can find the absorbance versus temperature curve of the other.
  • the same principle can be applied to composite curves for heteroduplex PCR- amplified products.
  • the method of the present invention has applications in diverse fields, including systematics and population studies, medical genetics, biodiversity assays, microbial pathology, forensic science and thermal characterisation of microsatellite allomorphs to determine relatedness. Detection of single-base changes using the method of the present invention would facilitate rapid identification of previously characterised mutations simplifying the identification of population-specific allotypes. Point mutations are also responsible for "carrier" status in many genetic diseases.
  • the automated detection of allotypes using allele-specific PCR-amplification and the method of the present invention offers an alternative to more labour-intensive electrophoretic techniques used for screening. Another area in which the method of the present invention has immediate application is HLA typing.
  • a method of comparing DNA including: providing a source of DNA for comparison; subjecting the source of DNA to a pre-determined temperature sufficient to cause a portion of the DNA to denature or to renature; determining the optical density of the DNA at the pre-determined temperature; and comparing the optical density of the DNA to the optical density of another source of DNA which has undergone similar temperature conditions.
  • Individual samples can be measured at a pre-determined denaturing or renaturing temperature and the optical density, will be dependent on the complex intermolecular and intramolecular interactions governed by the composition and order of nucleotides within the dsDNA. Small changes in the nucleotide sequence will affect the denaturing or renaturing of the DNA which will be reflected in the optical density by virtue of the proportion of the DNA denatured or renatured at a particular temperature.
  • Similar temperature conditions include conditions of concentration, temperature, and processing which are comparable or can be standardized so that a direct comparison between samples can be made.
  • the present invention provides a method of comparing DNA including: providing a source of DNA for comparison; subjecting the source of DNA to one or more temperatures; determining the optical density of the DNA at one or more temperatures to provide a denaturing profile; comparing the denaturing profile of the DNA to the denaturing profile of another source of DNA which has undergone similar temperatures.
  • Specific applications for comparing DNA include detecting single base substitutors between mutant and wild-type strains; detecting mutants in general by measuring subtle changes in T m ; conducting phylogenetic analysis for determining different strains such as viral strains; detecting micro satellite allomorphs for comparing relatedness; HLA typing; use of genetic tags; detecting polymorphic DNA; considering biodiversity and using mitochondrial PCR-amplified products to distinguish populations (Norman et al 1994).
  • This latter method also could replace sequence-specific oligonucleotide (SSO) probes currently used where population-specific markers are used (see Stoneking et al 1991). It could also replace allele-specific oligonucleotide (ASO) probes for detecting disease-specific mutations (Irvanson and Taylor 1991). Using this technique, rapid diagnosis facilitating appropriate application of therapeutic agents would be available Mack et al (1990). This method could readily facilitate population analysis to determine genetic distance and/or genetic diversity within and between populations. Genetic diseases could be efficiently screened for where parental phenotypes might predispose offspring to a particular condition, also allowing reliable chorionic villi sampling (CVS) to be carried out with a short time between sampling and the retrieval of results.
  • SSO sequence-specific oligonucleotide
  • ASO allele-specific oligonucleotide
  • the denaturing profile may be a curve of absorbance change versus temperature or it may be a standardised curve of ⁇ Abs/ ⁇ temperature Vs. temperature.
  • the curve may indicate denaturing or renaturing temperatures at points where there is a measurable change in absorbance.
  • the change in absorbance may be a sudden increase in absorbance indicating a melting domain.
  • the change is determined by a measurable deviation from the slope of the curve when there is no denaturation of the DNA.
  • the absolute measurable change in absorbance will be depend by the accuracy of the equipment used. Preferably the change will be greater than 0.01 unit absorbance/°C and most preferably the change will be greater than 0.1 unit absorbance/°C.
  • the denaturation profile may also be used to determine the length of the melting domains. Other specific uses of the method of the present invention include: 1. Detecting single-base substitutions
  • mtDNA Mitochondrial DNA
  • RFLP restriction digests of mtDNA
  • the method of the present invention can be used in a similar manner; the two characteristics, melting-domain length and domain-melting temperature can be used to gather unrefined phylogenetic information.
  • the method of the present invention is not constrained by the recognition of a specific sequence to be informative of DNA-sequence differences.
  • HLA-typing Histocompatability locus antigen (HLA)-typing has dramatically improved with the advent of PCR technology. HLA-typing is done by amplification of variable loci in the HLA gene complex followed by restriction digests using enzyme producing fragments that are then separated using gel-electrophoresis. The digest pattern is HLA-type-specific. It is possible for HLA-matches between donor and recipient - in the case of organ-transplants - to be confirmed or negated within 2-3 hours. Computerised storage of thermal denaturation profiles (TDPs) of donor HLA types would allow donor-recipient matches following PCR amplification of the recipients HLA loci without restriction digests or gel electrophoresis.
  • TDPs thermal denaturation profiles
  • a denaturation profile can be obtained in less than 1 hour. If donor-HLA TDPs can be stored on computer then easy comparison between HLA-TDPs with the HLA-TDPs stored and constantly updated at a central location, would facilitate rapid donor-recipient matches unimpeded by the delay imposed by electrophoresis. 6. Thermal denaturation profiles used with genetic tags.
  • Genetic tags are population-specific sequences usually from maternally- inherited mtDNA that can be used instead of physical tags to distinguish individuals from different source populations. Their practicality exceeds physical tagging systems in that a small tissue or blood sample is all that is required to uniquely identify the population of origin of the sample. They are unaffected by environmental factors and can be identified from tissue or remains where mtDNA can be extracted and amplified - using PCR. In the past either DNA- sequencing or RFLP data has been used to source DNA where the antecedents for genetic tags were known.
  • the method of the present invention does not require electrophoretic resolution of restriction fragments or the technical expertise required for DNA sequencing, After screening extant populations to identify fixed DNA-sequence differences that are population- specific, TDPs for PCR-amplified "tag" product can be used to identify the population of origin of an individual.
  • the method of the present invention can characterise many PCR-amplified "tags" simultaneously with reduced manipulation.
  • First derivative curves from the method of the present invention can be used to detect the thermal-fingerprint of a particular PCR-amplified DNA sequence where other dsDNAs are also present.
  • two highly conserved - class-specific - regions flanking a species-specific variable region - in soil bacteria or aquatic microorganisms - could be used to design PCR- primers that amplify the intervening species-specific variable region.
  • the amplified region would represent one or at most two melting domains, PCR- amplification of a tRNA gene would satisfy both criteria.
  • a series of peaks from first derivative curves, each corresponding to a PCR product-specific domain melting event peculiar to the species of origin will be seen.
  • Microbiological analysis using TDPs Thermal denaturation profiles of PCR-amplified dsDNA of strain-specific-
  • DNA sequences and genes coding for antibiotic resistance can be used as references to diagnose bacterial infections and bacterial-specific-therapeutic resistance.
  • XL-PCR the amplification of PCR-products as long as 30 kb has been possible
  • the ability of the method of the present invention to characterise long dsDNA molecules with a defined length means that large portions of bacterial genomes can be screened for polymorphisms, making this method an attractive alternative to the labour intensive isolation of base-changes - that result in novel antibiotic-resistant mutants - using genome- subtraction or DNA-DNA-hybridisation methods.
  • an apparatus for characterizing DNA including: a sample chamber for receiving a sample of DNA; a temperature source for applying one or more temperatures to the sample chamber such that the DNA is subjected to one or more temperatures; a detector for detecting optical density of the DNA sample; such that when a sample of DNA is subjected to one or more temperatures, the detector determines a change in optical density as the DNA denatures.
  • a computer system which analyses the optical density change preferably to further determine the denaturation temperature and the length of the melting domain.
  • the method may be automated by adopting a spectrophotometer with a heating block for a specific application; automatically increasing temperature, with continuous concurrent readouts of temperature and absorbance. This may produce an easily-interpreted and unique thermal denaturation profile (TDP) for a DNA sample preferably a PCR-amplified DNA of specific sequence and length.
  • TDP thermal denaturation profile
  • TRP thermal renaturation profile
  • Figure 1 shows a hypothetical plot of absorbance against temperature for a hypothetical PCR-amplified product with domain melting temperatures of 63°C and 78°C, and with a length of 2000 bp.
  • Figure 2 is a standardised plot showing the slope of the curve in Figure 1 for the hypothetical PCR amplified product showing two melting-domains.
  • Figure 3 shows a composite of the plots in Figures 1 and 2 with the second abscissa showing the length of the melting-domain.
  • Figure 4 shows the curve of A260 nm Vs. Temperature for denaturation of a 1050 bp Mitochondrial Cytochrome b gene (Cyt b) of Rhytidnoponera Species 12.
  • Figure 5 shows the curve produced by standardising A260 nm data from Figure 4 to the range of 0 to 1.
  • Figure 6 shows the curve of A260 nm Vs. Temperature for denaturation of a Hind Ill-digested lambda-bacteriophage between the temperatures of 82°C and 88°C.
  • Figure 7 shows the curve produced by the first derivative of the curve in Figure 6.
  • Figure 8 illustrates a schematic diagram for a spectrophotometer connected to a computer facility for automation of the STOP DNA analysis.
  • the calculations used to standardise this curve and thus determine melting-domain length are shown below. Two assumption are made; 1 , that the length of the PCR product is known and that the sample contains only 1 amplified dsDNA species and 2, domain-melting occurs between two consecutive data collection points (in the example data collection points are at 0.1 degrees Celcius intervals).
  • Figure 10 shows absorbance versus temperature curve after subtracting A ds .
  • Figure 11 shows the standardise curve of Absorbance versus temperature.
  • the curve in Figure 2 is transformed by multiplying all product- denaturation-specific absorbance data values for a specific denaturation curve by 1/A SS for that sample as in equation 11.
  • Figure 12 shows first derivative curve of a standardised plot for the denaturation of the same hypothetical 2kb PCR-product.
  • the peaks represent specific domain-melting events, the x-coordinate at peak-maxima corresponds to the temperature at which the rate of denaturation is greatest; the T m the melting-domain that denatures over this narrow temperature range.
  • Figure 13 shows the second derivative plot of the same, hypothetical 2kb PCR-product the x-axis intercepts labelled with " ⁇ " correspond to the x- coordinate at peak-maxima seen in Figure 4, or the T m of that particular melting-domain.
  • Example 1 - Characterisation of DNA The present invention defines a method of characterising DNA by detecting base changes to measure length of melting domains [l s ] and to determine denaturing temperatures [TJ.
  • the method includes providing a source of DNA; and measuring the change in optical density of the DNA at varying temperatures.
  • Ass Absorbance of the DNA when it is fully denatured (absorbance units)
  • Asam p ie measured absorbance of sample (absorbance units)
  • Astn standardised absorbance of sample at denaturation (absorbance units) All absorbance values taken at wavelength 260 nm while temperature increases
  • a correction factor; R 0 compensates for variation in concentration between samples. Using one of the samples as the standard, the factor R 0 is defined as
  • absorbance (A sa m p i e ) versus temperature (T) plots can be standardised. Using the formula for absorbance as a function of temperature
  • the range of the absorbance for the sample should be between 0 and 1. This is achieved by settling the absorbance value(s) for each sample before denaturation to zero as follows.
  • the denaturation temperature (T m ) of specific melting domains may then be calculated to indicate the temperature corresponding to the maximum rate of denaturation for that melting domain.
  • the plot of the first derivative of standardised absorbance versus temperature data reveals a series of peaks.
  • Figure 1 shows a hypothetical plot of absorbance against temperature for a hypothetical PCR-amplified product with domain melting temperatures of 63°C and 78°C, and with a length of 2000 bp.
  • the plot in figure 2 is of the slope of the curve in figure 1, and figure 3 shows is a composite of the plots in figures 1 and 2, with the second ordinate (y) axis showing the length of the domains.
  • the calculations below give the equations used to determine the length of domains, l s1 and l s2 . ln Figure 1 , it can be seen that three plateaux occur, labelled A, B and C, representing absorbance values under the following conditions:
  • T ml 63°C
  • T m2 78°C
  • a ds 0.33 (absorbance units)
  • a ss 0.50 (absorbance units)
  • a 1050 bp PCR product containing the mitochondrial cytochrome b. gene (cyt b.) of Rhytidnoponera species 12 was prepared using standard PCR techniques. A 100 ⁇ l PCR of the sample was purified using a Wizard-PCR ® p column supplied by Promega Corp.
  • Figure 4 shows the curve of A260 versus temperature for denaturation of the 1050 bp PCR product.
  • Table 1 Melting Domain Lengths and corresponding denaturation temperatures or a 1050 bp PCR product from Rhytidnoponera species 12.
  • FIG. 6 illustrates a plot of A260 versus temperature between 82°C and 88°C for the denaturation of the Hind Ill- digested lambda-bacteriophage.
  • Figure 7 shows the first derivative of the plot obtained in Figure 6 and illustrates a plot of ⁇ -A260/ ⁇ -temperature versus temperature (the first derivative) between 82°C and 88°C for the denaturation of Hind Ill-digested lambda-bacteriophage. This curve illustrates the resolution possible by using STOP DNA to determine denaturation temperatures for melting domains in large double-stranded DNA molecules.
  • each rise in absorbance represents the melting of a paricular domain.
  • a rise is followed by a plateau before the next domain-melting event occurs.
  • the ⁇ Abs/ ⁇ temperature versus temperature curve shows a set of peaks which correspond to the rises seen in the absorbance versus temperature curve while the zero or minima correspond to the plateaux. It is the x-coordinate's (temperature) value at the peak maxima on the ⁇ Abs versus ⁇ temperature curve that represents the greatest rate of melting of a specific domain designated herein as the melting temperature (Tm) of that domain.
  • Example 4 Automation of the STOP DNA analysis
  • the present method of characterising DNA based on changes in optical density have many advantages over the prior art. Its use requires little technical expertise, significantly reduces handling of samples and generates rapid results which are reproducible and reliable. To facilitate the rapid generation of results, automation of the method is relatively simple.
  • a spectrophotometer with an apparatus for automatically increasing the temperature can be connected to a computer to provide continuous concurrent readouts of temperature and absorbance.
  • FIG. 8 provides a schematic illustration of a spectrophotometer with labels and computer specifications which can be applied to the present method.
  • sample specification sample name, position in microtitre tray; AH, 1-12, select position (cuvette number) in sample cuvette numbers 1-96 (cell numbers
  • Ramp rate for heating samples Range from 0.5 to 5 °C/min.
  • the feedback for thermo-electric heated fan is the average of 40 consecutive readings from the four thermistors immersed in an equal volume of buffer identical to the sample buffer in the carousel cartridge at carousel positions l,26, 51 , 76. A new afferent temperature point is generated at every 1/4 turn of the carousel. These temperature points are collected and averaged over 10 revolutions. The average is used as the positive feedback temperature to set the gain for the thermo-electric heating fan. For example, using a centrifugal velocity of 60 rpm and a temperature ramp rate of 1°C/min each cell passes through the light-beam at approximately 0.016 °C intervals (1 time per sec). The feedback temperature, calculated as the average of all four probes over 10 cycles will be approximately +0.16°C/sec.
  • Data points are then stored in data arrays as (A260, Temperature) values every 10 cycles for each sample cell .
  • First derivative data can be calculated for each sample from these stored values as:
  • Data can be plotted as A260 versus temperature or for first derivative ⁇ A260/ ⁇ T versus temperature in the result field. Plots for individual samples can be recalled using their identifier code.
  • STOP DNA polymorphic DNAs
  • Figure 8 illustrates a schematic diagram for a spectrophotometer with labels and computer specifications which can be used in the present invention.
  • N Thermistor wire to transducer chip (power pick-up for the chip is through carbon brush contacts to conductors that encircle the base chassis as shown in the schematic). Output is digitised and transmitted to receiver (S).
  • S Received Signal
  • a sample of dsDNA of known length in aqueous solution is subjected to a slow increase in temperature.
  • the DNA denatures in sequence-specific blocks (these are known as melting-domains), producing a quantifiable change in the amount of light that is absorbed at wavelength 260nm.
  • the absorbance (A 260 ) and the temperature of the sample are recorded as the temperature increases.
  • the temperature difference between the melting temperatures of two analagous melting-domains - in a dsDNA-product of known length where specific base changes have resulted in an altered melting temperature of the melting-domain containing the mutation - may be small.
  • the smallest data increment temperature must be less than 0.5x the difference between the melting temperatures of the mutant and reference molecule.
  • Current methods (DGGE and TGGE) can detect differences as small as 0.5°C the method of the current invention should be able to detect differences as small as 0.008°C depending on carousel speed and rate of a temperature increase.

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Abstract

La présente invention décrit de manière générale un procédé de caractérisation de l'ADN. Ladite invention se propose en particulier d'examiner un procédé de détection des modifications des bases dans l'ADN en déterminant la densité optique à différentes températures. Le procédé utilise en particulier les densités optiques différentielles de l'ADN à un seul brin et de l'ADN à double brin de manière à caractériser de préférence la longueur, les températures de dénaturation et de renaturation des domaines de fusion. Le procédé de caractérisation comporte donc la fourniture d'une source d'ADN, la soumission de l'ADN à différentes températures, suffisantes pour provoquer la dénaturation et la renaturation d'une partie de l'ADN et la détermination de la densité optique de l'ADN dans ladite plage de températures.
PCT/AU1997/000595 1996-09-12 1997-09-12 Methode d'analyse d'un adn WO1998011433A1 (fr)

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AU41052/97A AU4105297A (en) 1996-09-12 1997-09-12 Method for analysis of dna

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AUPO2295A AUPO229596A0 (en) 1996-09-12 1996-09-12 Method for analysis of DNA

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Cited By (2)

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EP1207210A1 (fr) * 2000-11-15 2002-05-22 Roche Diagnostics GmbH Procédé d'analyse de produits de la PCR répétitive au moyen de la courbe de dénaturation thermique
US6664064B1 (en) * 2000-11-15 2003-12-16 Roche Diagnostics Corporation Method for melting curve analysis of repetitive PCR products

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WO1996009532A1 (fr) * 1994-09-22 1996-03-28 Abbott Laboratories Procede utilisant un guide d'ondes optique pour detecter des evenements de liaison specifique par diffusion de la lumiere
EP0711840A2 (fr) * 1994-11-09 1996-05-15 Hitachi, Ltd. Procédé et dispositif pour l'analysis d'ADN

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