MXPA97009706A - Method of and apparatus for diagnostic test of - Google Patents

Abstract

The present invention relates to an assay for detecting mutations in genes by a two-stage multiplex polymerase chain reaction amplification, followed by preferably preferably two-dimensional electrophoretic separation of the fragments based on both their size and their sequence of pairs of bas

Description

DR METHOD AND APPARATUS FOR DNA DIAGNOSTIC TEST The present invention relates to the diagnostic assay of DNA using polymerase chain reaction (PCR) amplification followed by electrophoretic separation of the resulting fragments to detect possible gene variants of mutational defects and the like; it relates more particularly to new and improved PCR techniques in combination with the preferably two-dimensional electrophoretic separation in denaturing gradient gels. The invention relates particularly to assays for the presence of DNA mutations in patients with hereditary diseases, including birth defects (eg, cystic fibrosis) and genetic predispositions to chronic diseases of adults (eg, cancer). More specifically, the invention relates to the rapid preparation of gene fragments and their subsequent efficient and accurate examination to detect mutations by a novel polymerase chain reaction (PCR) amplification. BACKGROUND OF THE INVENTION Genes with mutational defects (gene variants or alleles) can be identified by diagnostic DNA assay. Gene variants can be transmitted from parents to children. Some gene variants have a very powerful effect and are capable of causing disease on their own. Examples are many mutational variants of the transmembrane conductance regulator gene of cystic fibrosis that cause cystic fibrosis. Other gene variants act in combination with gene variants from other places. Examples include many common diseases (polygenic), such as heart disease and cancer. The assay is possible to detect genetic defects in a temperamental stage; that is, in embryo cells (pre-natal test), but also in a much later stage in young, adult and old individuals. Through the DNA diagnostic test you get information about a disease, sometimes before it has manifested. This greatly facilitates the management of the disease, for example prevention, treatment. For example, testing for the presence of gene variants in cancers or agents of infectious diseases is possible in order to predict, for example, the course of the disease, the response to therapy. It is also possible to test in individuals to know if they have a particular gene variant that they do not want to be transmitted to their descendants (carrier test). Finally, it is possible to test individuals at any time (from pre-natal to advanced age) to detect hereditary predispositions to diseases encoded in the genes. An example of one end of the spectrum is cystic fibrosis, for which prenatal testing and carrier screening has already become relatively common. The other end of the spectrum involves diseases that occur later, such as cancers and neurodegenerative diseases. The DNA diagnostic assay involves the analysis of the sequence integrity of individual genes. At present, this is expensive because a precise test requires sequencing, or decoding, the gene, which involves a lot of labor. To date, a standardized system for universally applicable and economical DNA diagnosis has not been made available (for a review, see Cotton, 1993, Current mutation detection methods, Mutat, Res. 285, 125-144). In order to be economical and widely accepted, a DNA diagnostic system must be precise (more than 95%), highly resilient, and labor-free. General background of analysis techniques Initially it is appropriate to review the general techniques involved in PCR amplification and electrophoretic separation of fragments, and where the technique has been applied and is currently applied. A sample of cells, such as derived from blood, is first chemically and physically treated to extract DNA strands carrying genes that occupy only about two percent of the total DNA material in a cellular genome, with the rest of the DNA material only messily fills the bottom. Although each cell has two copies of each gene, and such can be identified by blocks of composition or pairs of bases identified by sequences of letters A, C, G and T as a code of letters, they constitute such a tiny part of the long strings of DNA, which must be amplified by making many copies of them to allow inspection. This is carried out by thermal separation or denaturation of the DNA strand pairs, by mixing with appropriate primers to ligate or weld at the beginning and end of the gene fragments (eg, exons or gene coding regions) which should be investigated, which will be discussed in more detail later, and adding enough blocks of composition to generate copies of the exons of the genes. The successive repetition of this cycle of steps effects a cumulative copying of the exons to produce a purified and amplified quantity - a process which is generally referred to as polymerase chain reaction amplification or PCR amplification, mentioned above - and which is reviewed with more extensive, for example, in Molecular Pathology, Hein and Silverman, Carolina Academic Press, 1994, Chapter 2, Molecular Techniques and Their Automation in the Clinical Laboratory, pages 5-31 (inn-Deen). By now it is appropriate to inspect or analyze gene exons to determine if there are abnormal mutations. This is generally done by electrophoretic separation of the purified DNA fragments, preferably on the basis of both the size and sequence of base pairs, as described more fully by the co-applicant Vijg and A.G. Ultterlinden in Two-dimensional DNA typing: A Parallel Approach to Genome Analysis, Eills Horwood, 1994, in particular pages 33-40. Electrophoresis has not only been used for the separation of DNA fragments, but also for the separation of other substances that are not genes, such as, for example, protein analysis, as described in Electrophoresis 1993, 14, 1091-1198 . Machines are provided for the one-dimensional separation of DNA by electrophoresis with fluorescent dye labeling, such as the ABI Prism 377 DNA sequencer of the Perkin Elmer brand.; and for bi-dimensional DNA typing, as described by co-applicant Vijg and E. Mullaart et al. in Nature, 365, September 30, 1993, Parallel genome analysis by two-dimensional DNA typing, pages 469-471, in which an apparatus of the company Ingeny B.V. is described. from the Netherlands (Holland). The application of the electric field in first dimension (let's say horizontal) to an appropriate gel matrix (to be discussed later) in which the purified DNA fragments were introduced causes the separation of the fragments according to their size, in which the Larger particles move more slowly than smaller particles. By applying the electric field in an orthogonal direction (vertically) with a chemical gradient, such as urea / formamide disposed in the gel at successively higher concentrations, or with a thermal gradient established under the same considerations, the DNA fragments will migrate (now vertically) until they melt and settle in the gel matrix at particular vertical locations determined by the sequence. To prevent the whole of the DNA fragments from being founded. The latter can be adhered (before the electrophoresis process) to a quantity of only Gs and Cs, which are more resistant to fusion than the As and Ts. This so-called GC clamp effectively fixes each fragment in a position that is determined entirely by the sequence of the exon part of the fragments. If this sequence is modified in a single position, for example, by replacing an AT pair with a GC pair, it will melt later or earlier and will therefore be fixed in a different vertical position than the normal reference fragment. Such two-dimensional gene screening (TDGS) has the potential to become an economically accepted and widely accepted DNA diagnostic system. In this system, as described above, a large number of DNA fragments obtained by polymerase chain reaction (PCR) amplification from a specific sample of DNA are electrophoretically separated on the basis of both size and a sequence of base pairs. This system is highly accurate (specifically 99%) by virtue of the fact that the separation in the second dimension is based on gel electrophoresis with a denaturing gradient (DGGE). In fact, DGGE (gel electrophoresis with denaturing gradient) is the only system with such a degree of accuracy (Sheffield et al., 1993, The sensitivity of single-strand conformation polymorphism analysis for the detection of single base susbstitutions, Genomics 16 , 325-332, Grompe, 1993, The rapid detection of unknown mutations in nucleic acids, Nature Genet, 5, 111-117, Guldberg et al., 1993, Molecular analysis of phyto-lingonuria in Deanmark: 99% of the mutations detected by denaturing gradient gel electrophoresis, Genomics 17, 141-146). The automatic instrumentation for TDGS (bi-dimensional gene exploration) is already partly available and partly in development. The TDGS (bi-dimensional gene exploration) allows the detection of all possible mutations in DNA fragments obtained from one or more genes simultaneously with high performance and with a minimum of manual interference. One of the biggest obstacles to the widespread application of TDGS (two-dimensional gene exploration) in DNA diagnostics lies in the difficulty of amplifying many fragments simultaneously in the same reaction specimen by PCR (polymerase chain reaction) (PCR) multipiex). In fact, it is often not possible to find PCR primer sites (polymerase chain reaction) that amplify the relevant gene fragments and that simultaneously satisfy the requirements for both PCR (polymerase chain reaction) and gel electrophoresis. denaturing gradient, that is, allowing optimal PCR reactions and optimal fusion behavior of the amplified fragments. The current procedure begins with amplification by PCR (polymerase chain reaction) of target DNA regions, usually the regions of a gene that code for protein (exons). These amplification reactions are conducted separately, for example, if 27 exons are being analyzed in a gene, then it is necessary to perform 27 separate PCR reactions. In practice it is usually possible to conduct a few PCR reactions together in a test piece (eg, Edwards and Gibbs, 1994, Multiplex PCR: advantages, developmente and application, PCR Methods and Applications 3, S65-S75). It is clear that when testing a large number of individuals and when testing more than one gene simultaneously in the same TDGS (two-dimensional gene screening), the total number of pipetting steps and individual reactions there are what to accomplish can become very big. This increases the intensity of the trial workforce, but also makes it more complicated with a higher degree of possibility of human error. In fact, by virtue of this complexity, not even a complete laboratory automation in which all the pipetting steps are done automatically will solve this problem. The problem of not being able to simultaneously PCR amplify multiple fragments under identical reaction conditions in the same specimen is a major technical obstacle facing TDGS (bi-dimensional gene exploration) to meet clinical trial criteria, specifically ease of management in the laboratory. To reduce the number of PCR reactions by multiplexing, that is, conducting several PCR reactions in a reaction using multiple sets of primers is a non-trivial development. Current approaches for multiplexing are sometimes as simple as combining a few sets of primers for which the reaction conditions were determined separately. However, in the majority of cases multiplex PCRs must be developed with careful consideration of the regions to be amplified, the relative size of the fragments, the dynamics of the primers and the optimization of the experimental conditions of the PCR to accommodate multiple fragments. A key problem is the location of the primers. For the diagnosis of genes, it is generally sought to amplify the sequences of exons, the cleavage sites and regulatory regions. Primers for exon amplification PCR reactions are ideally placed in intron sequences adjacent to the exons. This provides some scope for adjusting the length of the fragment or the quality of the amplification, as well as information about mutations that affect the cut sites. These are the first limits to the selection of primers. Then, the primers should be placed in such a way that non-specific amplification does not occur in other sites that are not of the target sequences. In fact, the human genome has a length of 3 x 10 base pairs, which provides ample opportunity for fortuitous homologation of sequences between the target sequences and other non-target sequences. This problem is not typical for multiplexing, but it can also occur when you want to amplify only one fragment at a time. This is the second limitation to the selection of primers. For multiplexing, the primers should be selected so that their predicted hybridization kinetics are similar to those of other primers in the multiplexing reaction. This is a third limitation to the selection of primers. These limitations on the selection of primers, with all of the genomic DNA as a template, are the reasons why the multiplexing groups are usually small (typically less than 5 fragments). For an optimal separation in TDGS (two-dimensional gene exploration) there is a fourth formidable limitation to the selection of primers. TDGS (bi-dimensional gene exploration) requires DNA fragments of 100-600 bp (base pairs) on average. One of the two primers must be coupled to a GC-rich fragment to provide a GC clamp as the highest fusion domain in the fragment to be generated by PCR. This is essential to ensure the highest sensitivity to detect mutations in the second-dimensional gel (denaturation gradient) (Myers et al., 1985, Almost all substitutions of individual bases in DNA fragments attached to a GC clamp can be detected by denaturing gradient gel electrophoresis, Nuci. Acids Res. 13, 3131-3145: Myers et al., 1987, Detection and localization of individual base changes by denaturing gradient gel electrophoresis, Meth. Enzymol. , 155, 501-527). The primers should then be placed in such a manner that the target fragment comprises only a single single domain that is previously melted (at a lower concentration or temperature of urea / formamide) than the GC clamp that is attached thereto. It turned out that the optimal PCR conditions for both PCR (without even taking multiplexing into account) and optimal fusion profiles are difficult if not impossible to perform (for example, compare the RB DGGE design by Blanquet et al., 1993). , Identification of germline mutations in the RB1 gene by denaturing gradient gel electrophoresis and polymerase chain reaction direct sequencing, Hum Molec Genet 2, 975-979, with our present design). The present invention involves a combination of so-called long PCR with short PCR. Recently, PCR amplification methods were developed that allow the amplification of large fragments (up to 40 kb) of genomic DNA. We have taken advantage of this development using long PCR to first amplify all the coding regions of the target gene (s) into the smallest possible number of fragments. Using these long amplicons as a template, we then proceed to PCR amplify the small fragments, required for the TDGS (bi-dimensional gene exploration), in a multiplex format. In this way, the first target sequence is separated by amplification of the contaminating genomic DNA, which makes it possible to obtain the small PCR fragments under identical conditions of this pre-purified template. Using the above procedure, the primers selected solely on account of their optimal fusion performance of the PCR amplicons also exhibited optimal PCR behavior and even allowed extensive multiplexing. In part this phenomenon can be attributed to prepurification by long PCR. In fact, long PCR greatly increases the amounts of target sequence relative to other genomic DNA sequences, thereby greatly reducing the complexity of the reaction and increasing its specification. Even though the phenomenon described above can be explained by the reduction of complexity through the preparation of a purified mold, it is not possible to give an exact explanation. In fact, simply the magnitude of the effect is surprising and requires a re-evaluation of the factors involved in PCR optimization. However, there is one thing that is clear. The present invention alone makes it possible to design and perform a TDGS (efficient two-dimensional gene screening) assay, since now the primers can be selected based solely on their melting profile and multiplexing is greatly facilitated. The invention is also generally applicable. In fact, the selection of primers in each PCR-based diagnostic reaction is an important factor, and it turns out that many potential sites for priming provide unsatisfactory results. The multiplexing then generates additional problems, which is the reason why it is not widely used. The two-step system or stages of long PCR / short PCR amplification offers an immediate and simple solution to this problem.

OBJECTS OF THE INVENTION Accordingly, an object of the invention is to provide a new and improved method and apparatus for DNA diagnostic assay that obviates the difficulties described above. A further object is to provide novel detection of mutations in genes by multiplexing a two-step multiplex polymerase chain reaction amplification, using long and short PCR multiplexing followed by two-dimensional electrophoretic separation of the fragments based on both size and a sequence of base pairs. Other objects will be explained below and will be highlighted in connection with the appended claims.

BRIEF DESCRIPTION OF THE FIGURES Now we will proceed to explain the invention referring to the attached drawings, in which: Figure 1 illustrates the sequence of the steps of the process for carrying out this invention.

Figure 2 shows the fusion curves for exon 12 of RB with and without the GC clamp (specifically, retinal blastoma gene). Figure 3 shows maps of the tumor suppressor RB gene indicating the positions of the PCR primers for the long and short PCR reactions. Figure 4 shows a computer (computer) printout with the predicted positions of the short PCR fragments in a two-dimensional pattern of gel electrophoresis, with the specified specifications. Figure 5 shows the current separation pattern of the gel, indicating the correspondence with the theoretically predicted pattern. Figure 6 shows that with the long PCR product for exons 18-23 as a template, all 6 of the short PCR fragments (strips 7-12) are obtained, whereas with fully genomic DNA as a template, most of them are missing. the products (strips 1-6). It also shows that only 5 ng of fully genomic DNA is sufficient as a starting material for long PCR (strip 13), and that all short PCR products are obtained with the long PCR products as a template (strips 20-24 ). Figure 7 shows details of wild type fragments (normal homozygosis) and several heterozygous mutants.

SUMMARY OF THE INVENTION In summary, in an important aspect the invention encompasses a method for analyzing exons of predetermined genes derived from DNA that comprises adding pairs of primers to successive groups of the gene exons followed by carrying out the above reaction amplifications of polymerase chain in a common tube, as a first stage and a relatively long multiplex polymerase chain reaction; adding additional pairs of primers to each of the gene exons and carrying out polymerase chain reaction amplifications in a common specimen as a second stage and short multiplex polymerase chain reaction; and electrophoretically separating the gene fragments. The techniques and best preferred methods will now be described. DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention, as indicated above, involves the design of an accurate and efficient mutation detection assay based on a minimum number of two-stage multiplex PCR reactions, in combination with two-dimensional separation Automatic fragment detection to detect all possible mutations in the gene (s) simultaneously. Table 1 lists the different stages in the design of a TDGS (two-dimensional gene exploration) with the tumor suppressor RB (retinoblastoma) as a model. First, gene sequences are extracted from a database (for example, a gene bank), and target regions are defined, specifically exons, cutting sites, regulatory regions. Then, primers are placed to obtain all the target regions as the smallest possible number of fragments that can still be amplified through long PCR, that is, up to at least 20 kb (Case TaKaRa LA PCR. Product insertion). Some general guidelines have been described for selecting primer sequences for long PCR (Foord and Rose, 1994, Long-distance PCR, PCR Methods and Applications 3, S149-S161), but an empirical determination of the optimal primers is still necessary. Table 1. Design of a TDGS trial (two-dimensional exploration of genes). 1. Remove sequence from the database 2. Place primers so that the long PCR covers all desired regions (eg, coding sequences, cleavage sites, regulatory regions, probable sites of mutations) by the smallest possible number of amplicons. . Place primers for the short PCR in accordance with the following criteria: a) the desired target sequences should be covered by amplicons between 100 and 600 bp. b) the amplicons should have an optimal fusion behavior, ie, consist of a lower fusion domain in addition to the GC clamp attached to one of the primers. c) optimal distribution of the amplicons on the two-dimensional gel. d) similar reaction kinetics. . Set PCR conditions separately for each primer set with the long PCR products as template. . Develop multi-pllex amplification conditions by grouping sets of primers and adjusting the reaction components. As listed in Table 1, item 3, then, using the long PCR fragments as templates, the primers for short PCR are selected to yield fragments between 100 and 600 bp. The main selection criteria in this case is necessarily the fusion behavior of the fragments. In the ideal situation each amplicon should comprise only one fusion domain, which should be lower (less stable) than the GC clamp attached to it. The binding of a 30-40 bp GC clamp is achieved by making it part of one of the primers (Sheffield et al., 1989, The sensitivity of single-strand conformation polymorphism analysis for the detection of single base susbstitutions, Genomics 16, 325 - 332). The optimal fusion behavior is determined from each target sequence that is a candidate by the use of a computer (computer) program (for example, MELT87).; Lerman and Silverstein, 1987, Computational simulation of DNA melting and its application to denaturing gradient gel electrophoresis, Meth. Enzymol. 155, 482-501). An example of an amplicon with optimized fusion behavior through GC binding is shown in Figure 2 in connection with exon 12 for RB. In general, a collection of primers is selected that allow an optimal distribution, both in size and in DGGE dimension (gel electrophoresis with denaturation gradient), on the two-dimensional gel. Due to the high resolution of two-dimensional gels (size differences of 5-10 bp are easily resolved) this is generally not too difficult. In fact, with 50 fragments or less, the distribution of sites almost does not count and the primers can be selected simply according to their fusion behavior. Figure 3 shows the collection of amplicons selected for the RB gene, together with the long PCR fragments that served as templates. Together, the fragments of the short PCR represent more than 90% of the RB coding region. Figures 4 and 5 show the theoretical and empirical distribution of sites for the 24 exons of the RB gene covered by the amplicons shown in Figure 3. Although there are differences, most notably the site representing exon 11, our conclusion is that in total the fusion program accurately predicts the positions of the sites. It is important to understand that without the first long PCR step, the optimal fusion criterion is usually in conflict with other primer design criteria that are applied to PCR with fully genomic DNA as a template. In fact, for the RB gene it was found that it was impossible to select suitable conditions for both, an optimal separation in DGGE (gel electrophoresis with denaturation gradient) and optimal priming in PCR.fication stage that represents the long PCR apparently is a sine qua non condition for the design of an optimal set of PCR primers in TDGS (two-dimensional gene exploration). When the assay format is established, the two-step PCR amplifications are carried out in multiplex format. What represents the core of the present invention is the possibility of designing and carrying out multiplex PCR reactions. The need for the first long PCR stage for a successful multiplex PCR is demonstrated by the results shown in Figure 6. In Figure 6 the 6 belts on the left contain PCR products obtained after carrying out a multiplex PCR of exons 18-23 (6 fragments) of the RB gene, using different amounts of fully genomic DNA as a template. It is clear that products of the desired lengths are virtually not obtained. The latter was in contrast to strips 7-12, in which the products of the same multiplex PCR reaction were applied, but this time with the long PCR product as a template. The long PCR was carried out in different cycles and it is clear that only 5-10 cycles are required to generate enough mold for a successful multiplex PCR. Strips 13 through 19 contain the short PCR multiplex products obtained with the long PCR products as a template, at different amounts of starting material, ie different amounts of total genomic DNA used in the long PCR reaction. It is interesting to note that 5 ng of total genomic DNA is enough to obtain all the products. Because clinical material is not always available in abundant amounts (for example, breast cancer needle biopsies), this represents an important result, which indicates that successful trials with very small amounts of DNA can be carried out. Finally, strips 20-24 of Figure 6 contain the products of the 5 multiplex PCRs corresponding to the 6 sets of long PCR (the long PCR groups 1 and 6 were combined, see also figure 3 and table 2 to be discussed later) . Additional adjustments of the PCR and / or primer conditions should make it possible to obtain an even smaller amount of multiplex sets for this gene. In fact, there is no reason why the entire coding region of the RB gene can not be amplified in a single PCR reaction alone. After the second PCR, the fragments are allowed to undergo a complete round of denaturation / renaturation to facilitate the formation of heteroduplexes. A list of primer pairs for TDGS for the case of BR is presented in table 2; Exon numbers are listed in the table to the left, with long PCR primer codes for six exon groups (0 to 24-27) and short PCR primers for the 27 individual exons. Subsequent to PCR, the fragment mixture is subjected to two-dimensional electrophoresis in a denaturing gradient gel (FIG. 1). The availability of an automated instrument greatly simplifies this process. The instrument used here allows 10 gels to be handled simultaneously without manual interference, that is, to cut strips and load them into a second gel. All experiments involving optimization of experimental conditions were carried out using manual instruments. After the two-dimensional electrophoresis the gels are released from between the glass plates and stained with ethidium bromide or any other dye.

Table 2. Primer pairs for TDB of RB RB: Long PCR exons primers 5-31 size 0-2 TGTCAGGCCTGCCTGACAGACTTCTATTCAGCA 4.5 kb ATGTTAGCAGAGGTAAATTTCCTCTGGGTAATOO 3-6 GCAGTCATITCCCAACACCTCCCCTCTGT 9 kb AAGCCAAGCAGAGAATGAGGGAGGAGTACATTAC 23 Table 2 (cont ..) RB: Long PCR exons primers 5 '-3' size 11.07 TCAGCAGTTTCTCCCTCCAAOTCAGAGAGGC 10kb GAGACCAGAAGGAGCAAGATCAGGTAGTAG_12_-17 ACCATTCCCCCTACTCTCCATOOTCCATG 12.4 kb CTCACAGGAAAAATACACAGTATCCTOTTTGTGTGGC CCAGCCTTGCATTCTGGGGATGAAGC 18-23 14 24-27 kb AGTCGTAAATAGATTTTCTTCACCCCGCCCC GCCTTTGCCCTCCCTAAATATGGGCAATGG 7.3 kb CTGGGTTATCAGGACTCCCACTCTAGGGCC RB. PCR cut exon primers 5 '-3' size Tin Conj. (96UF) multiplex 2 (GCI) TTGATTTATAAGTATATGCCA 229 bp 30 E CAAAACGTTTTAAGAAAATCC 3 (GCI) CCAGTGTOTGAATTATTTAA 239 bp 27 A CCTITTATGGCAGAGGCTTATA 4 (GCI) GAATTGAAATATCTATGATT 270 bp 24A ATCAGAGTGTAACCCTAATA 5 (GCDTACTATGACTTCTAAATTACG 157 bp 27 A GTGAAAAATAACATTCTGTG 6 TGGAAAACTTTCTTTCAGTG 237 bp 17 A (GCI) GAATTTAGTCCAAAGGAATGC Table 2 (cont ..) RB: PCR long exons primers 5 '-3' size 7 (GCI) CCTGCGATTTTCTCTCATAC 257 bp 26 B GCAACTGCTGAATGAGAAAG 8 GTTCTTATCTAATTTACCACT 229 bp 27 B (GCI) TTTTAAAGAAATCATGAAGTT 9 (GCI) AGTCAAGAGATTAGATTTTG 227 bp 20 B ATCCTCCCTCCACAGTC 10 (GCI) GACATGTAAAGGATAATTOT 222 bp 21 B GCAAATCAATCAAATATACC 11 AGTATGTGAATGACTTCACT 174 bp 21 B (GCI) TATAATATAATTAAAAGTAGG RB PCR short (cont ..) exon primers 5 '-3' size Tin Conj. (96UF) multiplex 12 CTCCCTTCATTGCTTAACAC 211 bp 24 C (GCI) TTTCTTTGCCAAGATATTAC 13 (GCI) GATTACACAGTATCCTCGAC 224 bp 34 C GCAGTACCACGAATTACAATO 14 (GCI) GTGATTTTCTAAAATAGCAGG 179 bp 35 C ACCGCGCCCGGCTGAAAT 17 (GCDTTCTTTGTCTGATAATAAC 380 bp 26 C CTCTCACTAACAATAATTTGTT Table 2 (cont.) RB: Long PCR exons 5 '-3' size primers 18 (GCDGACTTTTAAATTGCCACTGT 393 bp 33 D ATTCCCTACAGTTTCTTTAT 19 (GCDCAACTTGAAATGAAGAC 248 bp 34 D CGTCCCGCTGCTCTTGAAAATAATCATC 20 (GCI) AAAATGACTAATTTTTCTTATTCCC 227 bp 44 D AGGAGAGAAGGTGAAGTGC 21 (GC2) CATTCTGACTACTTTTACATC 201 bp 28 D CGGGCTTACTATGGAAAATTAC 22 (GC3) CTTTTTACTGTTCTTCC 194 bp 33 D CCAATCAAAGGATACTTTTG 23 (GCI) TCTAATGGGTCCACC 281 bp 38 D CCCTACTTCCCTAAAGAGAAAAC 24 CGGAATGATGTATITATGCTCA 195 bp 22 E (CGI) TTCTTTTATACTTACAATOC 25 (GCI) ATGATTTAAAGTAAAGAATTCT 245 bp 38 E CATCTCAGCTACTGGAAAAC 26 (GCI) TCCATTTATAAATACACATG 161 bp 32 E ATTTCGTTTACACAAGGTG 27 (GCI) TACCCAGTACCATCAATGC 191 bp 43 E TCCAGAGGTGTACACAGTG GC Clamps: GCI: CGCCCGCCGCGCCCCGCGCCCGTCCCGCCC (30mer) GC2: CGCCCCGCGCCGCCGCCCCGCCCCCGCCCGTCCCGCCC (38mer) GC3: CGCCCCGCCGCGCCCCGCGCGCCCGGTCCCCGCGC (35mer) The resulting standards were documented and evaluated (by ocular and image analysis) to detect the occurrence of mutations. Under the applied conditions, specifically GC annealing and heteroduplex, heterozygous mutations result in 4 points, the 2 homoduplex variants and the 2 heteroduplex variants (illustrated in figure 7). The latter are not always separated. Since mutations can also occur in a homozygous state it may be necessary to mix each sample with a control sample before PCR, to ensure that heteroduplex molecules will be present. The following provides details of the ways in which the embodiments of the present invention can be made and used in order to achieve the accurate and efficient preparation and examination of gene fragments in diagnostic DNA assays. This description, which focuses on an exemplary gene or model, specifically the tumor suppressor RB gene previously discussed, should not be constructed in the sense that it limits the invention. The same procedure can be used in other genes and / or even more PCR fragments can be combined in the same specimen, within the possibility of one skilled in the art, and should be considered to fall within the scope of this invention. A. Design of the two-step RB assay TDGS PCR Obtaining the sequence: The sequence of the RB gene is taken from a database, specifically from a gene bank. The target regions are defined, that is, the exons, cutting sites, regulatory regions. Selection of primers for long mul tiplex PCR.

The primer pairs for the long PCR are placed in such a way that all the target regions are covered by the smallest possible number of fragments that can still be amplified by the long PCR. Long PCR rods are also selected for the highest specificity, optimal annealing temperature and minimal self-complementation for long multiplex PCR, for example by the use of software for primer design. The design of the multiplex long PCR is relatively simple in view of the broad field of positioning of the primers.

Selection of primers for short PCR. The primer pairs for the short PCR are selected on the basis of the following criteria: a) the desired target sequences should be covered by amplicons between 100 and 600 bp. b) the amplicons should have an optimal fusion behavior, ie, consist of a lower fusion domain in addition to the GC clamp attached to one of the primers. c) optimal distribution of the amplicons on the two-dimensional gel. d) similar reaction kinetics.

Criterion b) above is often in conflict with standard criteria for the design of primers, if applied to total genomic DNA. In fact, the present invention proved both necessary and sufficient for the design and performance of TDGS as a fast, accurate and practical tool for the detection of mutations in the RB gene. The multiplex groups are selected empirically based on the behavior of the primers in several multiplex reactions. For RB multiplex groups were made according to the long PCR. That is, all short PCRs with a long PCR fragment as a template were amplified together as a multiplex group. Currently, two long PCR groups were combined as a single multiplex group. All short PCR fragments can also be amplified together. As explained above, Table 2 lists the primer pairs used for the long and short PCRs, the fragment sizes, the annealing temperatures, the melting temperatures and the five different multiplex groups. B. PCR and heteroduplex reactions The primers (deprotected and desalinated) can be obtained from several sources. Our primers were obtained through Gibco BRL. For long-term storage, the primers should be maintained, for example, in a solution in one foot of 100 μM solution in ultrapurified water, at -20 ° C. For short-term use, we kept them at -20 ° C as a 12.5 μM solution in ultrapurified water. We carried out our PCR reactions in thermowell test tubes (Costar, Cambridge, MA) in a Gene E thermal cycler (Techno, Cambridge, MA) equipped with a heated lid, eliminating the need for an oil layer on the samples. The long multiplex PCR reactions (6 fragments) were carried out in a volume of 100 μl with 5-500 ng of genomic DNA as template and 0.2 μM of each primer, using the PCR LA kit (TaKaRa). PCR reactions are carried out according to the manufacturer's instructions. The conditions were as follows: First, a cycle of 94 ° C, 1 min., Followed by 30 cycles of 98 ° C, 20 sec / 68 ° C, 12 min. with increments of 10 s per cycle, and finally a cycle of 72 ° C, 12 min. The PCR products are stored at -20 ° C for later use. The short PCR reactions are carried out using the same Gene E thermocycler, in a volume of 50 μl with 2 μl of the long PCR product, 0.2-0.5 μM of each primer, 0.25 mM dNTPs, 2.5-4.5 mM MgCl2, 3 units of Taq polymerase (Gibco BRL or Promega). The conditions of the PCR are as follows: A cycle of 94 ° C, 2 min., Followed by 30 cycles of 94 ° C, 40 sec / 41 ° C, 40 sec / 69 ° C, 2 min (with increments of 2 s per cycle) and finally a cycle of 72 ° C, 10 min. After short PCR, the fragments are heteroduplexed by a complete round of denaturation / renaturation. Specifically 98 ° C, 10 min / 55 ° C, 30 min / 41 ° C, 30 min. After PCR and heteroduplexing, the contents of the specimens are mixed and 1/10 volume of loading buffer is added. Based on ethidium bromide staining there is usually enough sample for several trials. When the total volume is too large for the capacity of the slot, the sample must be precipitated with ethanol (before adding the buffer) and redissolved in a smaller volume. C. Two-dimensional electrophoresis The instruments for both manual and automatic bidimiensional electrophoresis were from the aforementioned company Ingeny B.V. (Leiden, Netherlands) . For manual electrophoresis, mixtures of the DNA fragments were first subjected to size separation using a 0.75 mm thick gel, 9% PAA at 45 ° C for 5-6 h. The separation pattern was visualized by staining with ethidium bromide for 10 min and transillumination of the gel by UV rays, gel that is on a glass plate to protect the DNA fragments from the UV light. The region of 100 to 600 bp in the central part of the strip (ie the edges are not included) was cut quickly and applied to a 9% PAA gel 1 mm thick that contained a 0-60% gradient (RB) or 30-90% (p53) urea / formamide (UF). The gradients were poured using a simple gradient former (Gibco BRL). Electrophoresis was carried out for 7.5-11 h at 60 ° C and 200 V. After electrophoresis the gels were stained with 0.5 μg / ml ethidium bromide for 15-20 min and stained in water for another 15 min. The patterns were documented under UV illumination using a Polaroid camera.

For automatic bidimiensional electrophoresis, gels were poured, ten at a time, into the gel-casting device that comes with the automated two-dimensional electrophoresis instrument according to the manufacturer's instructions (Ingeny B.V., Leiden, The Netherlands). After the polymerization, the gels (between the glass plates) are removed from the gel drain box and cleaned with damp cloth. They are then placed inside the instrument in accordance with the manufacturer's instructions, specifically on two gel cartridges with silicone side seals. The instrument containing the buffer, heated to 45 ° C, is put in the one-dimensional mode with the electric current turned off. After adding loading buffer, samples (up to 40 μl) are loaded into the V-shaped wells of the gels in the automated two-dimensional electrophoresis instrument. Gels were used with 9% acrylamide, 0.25% TAE, with a gradient of 0-60% urea / formamide. The first dimension is run at 180 V for 4 h at 45 ° C. The second dimension was run at 200 V for 7.5-11 h at 60 ° C. After electrophoresis, the gels were stained with ethidium bromide and the patterns were documented under UV illumination as described for manual instruments.

In sum, even when the present invention is described in connection with two-stage amplification (long and short) polymerase chain reaction multiplex, followed by two-dimensional electrophoretic separation, it is also useful with one-dimensional electrophoresis or other methods for the detection of mutations that require amplified PCR target sequences. Additional modifications will occur to those skilled in the art, and these are considered to fall within the scope and spirit of the invention, as defined in the appended claims.

Claims (1)

1. CLAIMS A method of analyzing predetermined exons of genes derived from DNA characterized by the fact that it comprises adding pairs of primers to successive groups of gene exons, followed by effecting amplification of their polymerase chain reaction in a common test tube, such as first stage and relatively long multiplex polymerase chain reaction; adding additional primer pairs to each of the gene exons and effecting polymerase chain reaction amplification thereof in a common probe as a second step and short multiplex polymerase chain reaction; and electrophoretically separating the gene fragments. A method according to claim 1, characterized in that the gene fragments are separated electrophoretically on the basis of their size along a dimension. A method according to claim 2, characterized in that the gene fragments are further subjected to additional electrophoretic separation of base pairs sequences along an orthogonal direction and along a gradient of thermal denaturation. or chemical to distribute the gene fragments at particular locations of sequence along the orthogonal dimension; and compare such locations with those of normal gene fragments to detect I genetic mutations. A method according to claim 3, characterized in that the primers are oligonucleotide primers labeled with fluorescent dyes. A method according to claim 3, characterized in that the nucleotide structural blocks used in the polymerase chain reaction are labeled in fluorescent form. A method according to claim 3, characterized in that the electrophoretic separation is carried out in a denaturing gradient gel. A method according to claim 6, characterized in that a gradient of urea and formaldehyde is applied in a gel to separate the fragments. A method according to claim 6, characterized in that a temperature gradient is applied in a gel to separate the fragments. A method according to claim 3, characterized by the fact that for the retinoblastoma gene the groups of exons of the long PCR genes are exons 1-2, 3-6, 7-11, 12-17 , 18-23 and 24-27. A method of analyzing predetermined exons of genes derived from DNA characterized by the fact that it comprises adding pairs of primers to successive groups of the gene exons, followed by carrying out amplification of their polymerase chain reaction in a common test tube, as a first stage and relatively long multiplex polymerase chain reaction; and adding additional primer pairs to each of the gene exons and performing polymerase chain reaction amplification thereof in the common coupon as a second step and short multiplex polymerase chain reaction.