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WO2008059578A1 - Procédé d'amplification pcr multiplex - Google Patents

Procédé d'amplification pcr multiplex Download PDF

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Publication number
WO2008059578A1
WO2008059578A1 PCT/JP2006/322898 JP2006322898W WO2008059578A1 WO 2008059578 A1 WO2008059578 A1 WO 2008059578A1 JP 2006322898 W JP2006322898 W JP 2006322898W WO 2008059578 A1 WO2008059578 A1 WO 2008059578A1
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primer
snp
annealing
minutes
reaction
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PCT/JP2006/322898
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English (en)
Japanese (ja)
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Tetsuya Tanabe
Nobuhiko Morimoto
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Olympus Corporation
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Publication of WO2008059578A1 publication Critical patent/WO2008059578A1/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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a multiplex PCR method.
  • the multiplex typing method has been proposed in the late 90s to meet the recent demands for lowering genotyping costs and higher throughput.
  • a microarray for SNP detection of cytochrome P450 protein from Roche is also more complex and more flexible in detection than a multiplex method in terms of reaction and detection in a single reaction vessel.
  • a method is proposed.
  • This method is widely used for genetic diagnosis and the like.
  • gene analysis is multiplexed using DCN. If the PCR that cuts around the SNP could be multiplexed in the same way, it would be desirable in terms of cost and work efficiency.
  • the key to such a multiplex typing method is an artificial sequence called a DNA tag, a reaction that converts a natural gene sequence into an artificial sequence, a probe shape for the reaction, and a DNA tag. It is the identification detection technology. Since multiple genes are converted into DNA tags in a one-to-one correspondence within the same solution and detected, each DNA tag does not cross-hybridize with each other so that it reacts independently. It is designed to have the same reaction force and melting temperature (Tm). Unlike microarrays, in which gene sequences themselves are probed using a detection device, they can be freely associated with genes, and if the same DNA tag is always detected at the detection stage, the same detection is possible even if the target gene changes.
  • Tm melting temperature
  • PCR product length force Set the primer spacing so that it is relatively short, about 3 ⁇ 400 base pairs.
  • Primers should be designed with a length of up to about 30 bases, which is inconvenient to synthesize.
  • an object of the present invention is to provide an efficient multiplex PCR method in which primer design is easy and the certainty of obtaining an amplification product is high.
  • the present inventors predicted that the cause of failure of the multiplex PCR method was cross-hybridization and variation in amplification efficiency.
  • the cross hive The focus was on suppression of redization, and variations in amplification efficiency were not considered. Therefore, focusing on this point, it is assumed that the main cause of "variation in amplification efficiency" is “variation in primer efficiency and irregularization efficiency", and a multiplex that suppresses variation in amplification efficiency.
  • the present invention is a multiplex PCR method
  • primers with a length of 30 bases or more Using primers with a length of 30 bases or more,
  • Annealing at least once in a thermal cycle 3 min. To 10 min.Including extension reaction, or annealing.Extension reaction time is extended to 3 min. To 10 min in the last thermal cycle according to the cycle. And
  • a multiplex PCR method is provided.
  • a PCR amplification method comprising the steps of:
  • the primer provides a method that is 30-60 bases in length.
  • the primer provides a method that is 32-50 bases in length.
  • the primer provides a method that is 35-45 bases in length.
  • the annealing / extension reaction provides a method that is performed to include at least one or more annealing period of 4 minutes or more during the thermal cycle.
  • the annealing / elongation reaction provides a method in which at least one annealing cycle of 6 to 10 minutes is performed in the thermal cycle.
  • the annealing temperature is based on the evaluation value (Q-Score) predicted by Visual OMP (DNA Software)! /, And the hybridization efficiency and prediction of an evaluation value (Q-Score) of 850 or more A method is provided that is less than or equal to the Tm of the primer to be produced.
  • FIG. 1A is a graph showing a prediction result of hybridization efficiency.
  • FIG. 1B is a graph showing a prediction result of hybridization efficiency.
  • FIG. 2 is an electrophoretogram showing the PCR results in a single plex.
  • FIG. 3 is an electrophoretogram showing the results of annealing temperature examination.
  • FIG. 4 is an electrophoretogram showing the results of examining the primer concentration.
  • FIG. 5 is an electrophoretogram showing the results of examination of the enzyme DMSO concentration.
  • FIG. 6 is an electrophoretogram showing the results of thermal cycle studies.
  • FIG. 7 is a schematic diagram showing an outline of a method used in Examples.
  • FIG. 8 is a scatter plot of array detection intensity showing the typing results for each SNP using the method of the present invention.
  • FIG. 9 is a scatter plot of array detection intensity showing the typing results for each SNP using the method of the present invention.
  • FIG. 10 is a scatter diagram of array detection intensities showing typing results for each SNP using the method of the present invention.
  • FIG. 11 is a diagram showing definitions of SNP typing results and QV values.
  • the multiplex PCR method of the present invention is characterized in that a primer longer than the primer length (for example, less than 30 bases) as in general common sense in ordinary PCR is used. It is considered that such primer design can make the primer hybridization efficiency uniform. At the same time, the primer specificity can be increased by designing the primer length longer. Therefore, even for a sample mixed with a long base such as 1000 bases, a multiplex PCR product can be obtained specifically by making the annealing efficiency uniform. In addition, even for a sample containing many complex or similar sequences such as the human genome, it is thought that the target product can be specifically amplified with the same amplification efficiency.
  • the length of the primer used in the multiplex PCR method of the present invention needs to be long enough to avoid cross-hybridization, and is longer than the range of general common sense.
  • the length of the primer is, for example, about 30 to 60 bases, preferably about 32 to 50 bases, and more preferably about 35 to 45 bases.
  • the Tm value of a normal primer is usually higher than that of a short primer.
  • the primers used in the multiplex PCR method of the present invention can be designed with various sequences depending on the vertical sequence to be amplified.
  • the primers used in the multiplex PCR method had to be designed so that the PCR product could be up to 300 bases or the length of the amplified product would be the same.
  • the length of is not limited. Therefore, the selection range of primer design is increased, and it is possible to design a primer set that was impossible with the conventional multiplex PCR method.
  • the primer predicts the secondary structure of the cage and the primer, makes the hybridization efficiency uniform, and provides a multiplex PCR product.
  • Such design, secondary structure prediction, and calculation of high hybridization efficiency can be performed using, for example, Visual OMP (DNA Software).
  • the optimal sequence can be selected by setting the design parameters as follows: primer length 30-45mer; primer design Tm 70 ° C to 100 ° C.
  • Other design parameters can be determined based on the results of analyzing the correlation between primer design parameters and PCR.
  • those skilled in the art will be able to easily design a primer having the long sequence as described above and capable of amplifying a desired saddle type.
  • a target nucleic acid amplification step is performed using the above primers.
  • nucleic acid means all DNA and RNA including cDNA, genomic DNA, synthetic DNA, mRNA, total RNA, hnRNA, and synthetic RNA.
  • the target nucleic acid to be detected or quantified can be any nucleic acid having an arbitrary sequence, but a nucleic acid that can serve as a disease marker, such as a gene causing a disease, a cancer-related gene, or a nucleic acid derived from a virus, Preferred target nucleic acids. Therefore, the sample includes body fluids such as blood, urine and saliva, but any sample other than body fluids can be used.
  • the sample may be dissolved in the liquid by an appropriate method such as enzyme treatment, addition of a surfactant or organic solvent.
  • the target nucleic acid can be arbitrarily changed in carrying out this method. For example, by preparing a large amount of human genomic DNA necessary for this method by culturing cells in large quantities and culturing them in large quantities, or by obtaining a large amount of peripheral blood, the genomic DNA force detection reaction can be started directly. Alternatively, a small amount of genomic DNA is obtained, and a detection reaction is detected from a sample that has amplified genomic DNA nonspecifically using the WGA method (Whole Genome Amplification) such as the reagent kit GenomiPhi of Amersham Biosciences. You may start.
  • WGA method Whole Genome Amplification
  • the detection reaction may be started from the amplification of a specific sequence using primers such as PCR, multiplex PCR, and asymmetric PCR.
  • primers such as PCR, multiplex PCR, and asymmetric PCR.
  • the target nucleic acid is assumed to be genomic DNA or the like. It may be amplified by such as.
  • the obtained sample is heated to 95 ° C and rapidly cooled to 4 ° C to form a single strand, or heated to 95 ° C in a solution with a very low salt concentration. Then, after performing fragmentation operations such as single-strand and fragmentation, such as fragmentation with ultrasound, fragmentation with restriction enzymes, and cleavage with restriction enzymes, perform detection.
  • the target nucleic acid amplification step first, the primer, the target nucleic acid, and an appropriate reaction solution are mixed and thermally denatured.
  • This step may be performed using general PCR conditions. For example, it is preferable to perform denaturation by heating at 94 ° C for 2 minutes before each cycle, and at 94 ° C for 30 seconds as the denaturation step for each cycle.
  • reaction solution used in the amplification step a general PCR reaction solution can be used, and a commercially available kit can also be used.
  • enzyme AccuPnme II (Invitrogen), noffer ⁇ : AccuPnme II master mix, filma ⁇ 0.1 ⁇ each, vertical DNA: 5 ng, reaction volume: 20 L be able to.
  • a primer is annealed to the single-stranded nucleic acid generated by heat denaturation.
  • the multiplex PCR method of the present invention is characterized by annealing at a temperature relatively lower than the Tm value of the primer designed as described above. In particular, it is preferable to perform annealing at a temperature that is significantly lower than conventionally considered. Thereby, the hybridization efficiency is increased, and it is considered that the hybridization efficiency of the probe can be increased for any of the saddle types. Therefore, in the conventional multiplex PCR method, fragments that have not been amplified can be amplified, and the amplification efficiency is increased.
  • the Tm value of a primer is a Tm value predicted by a method well known in the art, and is a Tm value predicted by, for example, Visual OMP (DNA Software).
  • the temperature is significantly lower than the Tm value of the primer even at a relatively high annealing temperature.
  • the annealing temperature is lowered too much, nonspecific annealing of the primer is likely to occur, and the amplification of the target sequence is inhibited by nonspecific amplification, but the primer used in the present invention is more than the conventional primer. It is considered that amplification efficiency is good because of sufficient forex annealing efficiency.
  • the annealing temperature is 68. Even at ° c, the temperature is sufficiently low.
  • the multiplex PCR method of the present invention is characterized in that an extension reaction is performed for a longer time compared to a normal extension time.
  • the extension reaction is, for example, 3 minutes, 4 minutes, 5 minutes, and 6 minutes to less than 10 minutes, 5 minutes to less than 8 minutes, preferably about 6 minutes. Therefore, it is preferable that the enzyme used in the multiplex PCR method of the present invention has long-lasting activity.
  • the primer used in the multiplex PCR method of the present invention since the primer used in the multiplex PCR method of the present invention is long in length, it has a relatively high annealing temperature. Therefore, when annealing is performed, the temperature may reach a temperature at which the extension reaction proceeds. In such a case, it is also possible to use shuttle PCR, in which both the annealing temperature and the extension reaction temperature are the same, so that both proceed simultaneously.
  • the multiplex PCR method of the present invention since the multiplex PCR method of the present invention has a long extension time, the amplification reaction can sufficiently proceed even if the amplification product is long. Therefore, even if the length of the amplification product is not the same as in the conventional multiplex PCR method, according to the multiplex status PCR method of the present invention, all the different amplification products can be amplified uniformly.
  • the multiplex PCR method of the present invention uses a primer longer than the length of the primer (eg, less than 30 bases) as in common general knowledge in normal PCR, and normal extension.
  • the reaction can be performed in the same process as the conventional PCR method, without being limited to the above-described step, except that the extension reaction is performed for a longer time compared to the time.
  • the step of heat denaturing a DNA strand and the step of synthesizing a complementary strand with a polymerase can be carried out under general reaction conditions depending on the type to be amplified.
  • the reaction conditions to be considered include, for example, the temperature and time of each step, the composition of the reaction solution, the concentration of constituents, and the like.
  • reaction conditions used can be those according to the seller's instructions if a commercially available enzyme is used. Those skilled in the art can easily select the temperature and time, conditions of the reaction mixture, the concentration of the components, etc., excluding the conditions peculiar to the multiplex PCR method of the present invention described above. Let's go.
  • the following modes are conceivable.
  • the following can be considered as uses and places of the detection method of the present invention.
  • the relationship between human genotype and disease There are research applications such as detection of drug sensitivity, detection of protein binding changes in the gene regulatory region, and molecular biological analysis of gene polymorphisms when the target organism is changed from human to another. These studies will be conducted in research institutes and laboratories such as universities and companies.
  • the relationship between the gene and a specific disease, risk of morbidity, and drug sensitivity are identified, testing to select treatment methods at the hospital's laboratory center and prevention at the clinical dock It can be used for medical purposes such as diagnosis and drug sensitivity testing for the selection of anticancer drugs with small side effects.
  • the user himself / herself carries out as a genetic polymorphism detection reagent kit for research and diagnosis for carrying out the method, and by an automatic reaction apparatus that automatically processes the method. It is possible to conduct a contract research on behalf of the subject or a diagnosis at a laboratory.
  • Example 1 Examination of SNP sequence amplification by multiplex PCR method
  • the SNP base sequence to be detected was obtained from the Japanese SNP database JSNP (http://snp.ims.u-tokyo.ac.jp/indexja.html) maintained by the Institute of Medical Science, the University of Tokyo. .
  • the respective accession numbers are IMS-JST164838 (SNP # 3), IMS-JST058048 (SNP # 4), IMS-JS T005689 (SNP # 5), IMS-JST054229 (SNP # 6), IMS-JST001164 (SNP # 7) ), IMS-JST017 558 (SNP # 8), IMS-JSTl 75404 (SNP # 9), IMS-JST054214 (SNP # 10), IMS-JST011815 (SNP # 11), IMS-JST156026 (SNP # 12) A total of 10 SNPs.
  • Table 2 shows the results of sequencing the samples using the Sanger method.
  • PRISM 3100 Genetic Analyzer from Applied Biosystems, and each of them using the Mitsui Information Development's sequencer output waveform analysis software wave flat. The SNP allele was determined.
  • the method of processing each genomic sample will be described in detail.
  • Visual OMP DNA Software
  • This software can calculate the hybridization efficiency of DNA by inputting DNA concentration, solution salt concentration, humidity, etc.
  • the hybridization efficiency at the PCR primer concentration in the PCR buffer was 30 to 40 mer primer length in order to make this value 100% for all primers.
  • secondary structure prediction is also performed on other design parameters, primers, and the template to be hybridized with the primer, and in particular, the selected primer sequence is predicted to be truly hybridized and more difficult. Primer sequences that have stable intramolecular structure and high hybridization efficiency were selected.
  • Fig. 1 shows the design results related to hybridization efficiency, which is an important guideline for the above-mentioned multiplex PCR primer design. In the MTL series with longer primer lengths, hybridization efficiency was expected to be close to 100% at any SNP site ( Figure 1 and Table 3).
  • the condition examination process is shown.
  • the condition study proceeded in the following order:
  • PCR was performed using a single plex, and it was confirmed that the primer was working. The result is shown in figure 2. Although amplification was confirmed in all 10 SNP sites, multiplex PCR was not possible under the same PCR conditions.
  • DMSO was also added at the same time. Amplification is improved by adding DMSO. There was a part where the amplification weakened with the addition of DMSNO, and it became clear that uniform amplification could not be obtained with DMSO.
  • Figure 3 shows the results of the annealing temperature study.
  • the result of the PCR reaction is greatly affected by the enzyme system. Therefore, the reaction was carried out using four types of enzymes, AmpliTaq Gold Master Mix (ABI), AccuPrime Super Mix I / II (Invitrogen), and HotStar Taq (Qiagen), and compared. In the AmpliTaq Gold Master Mix system, the effect was confirmed by changing the DMSO concentration at the same time. The results are shown in FIG.
  • extension time was sufficiently long using shuttle PCR, amplification was performed uniformly and strongly. Therefore, the extension time was determined to be 6 minutes, which is considered to be a sufficiently long extension time. Seven bands were visible on electrophoresis, and up to eight bands could be observed on the bioanalyzer. Forces with unresolved peaks Clearly unamplified SNPs are gone. (Resolution head
  • Example 2 SNP detection using multiplex PCR
  • Two probes shown in Fig. 1 are prepared to detect the target nucleic acid contained in the solution.
  • One is a common probe of an oligonucleotide containing a sequence complementary to a partial sequence (partial sequence) of a target nucleic acid and labeled with 3 'end such as piotin.
  • the label of the above-mentioned common probe may be any substance that can specifically bind to a specific substance such as piotin or an antibody that is not limited to piotin (for example, in the case of piotin, it is specific to streptavidin. Can be combined).
  • the other oligonucleotide has an artificially designed tag with a base sequence force of SD, D1 and ED at the 5 'end, is complementary to the sequence of a part of the target nucleic acid, and is A query probe having a sequence adjacent to the sequence complementary to the probe target.
  • the probe is referred to herein as a tag nucleic acid.
  • the artificially designed base sequence is arranged on the 5 ′ end side with respect to the complementary sequence.
  • the 5 'end of the sequence complementary to the target cDNA of the above common probe is phosphated.
  • the tag portion and the query probe portion of the tag nucleic acid may be oligonucleotides that are partially or entirely in a stranded state. At this time, construct a double-stranded oligonucleotide. The other strand formed is an oligonucleotide with a sequence complementary to the sequence of SD, 01 ⁇ and ED.
  • the query probe portion and the common probe of the tag nucleic acid can be arbitrarily designed for each target gene that has been detected to be present or absent in the solution.
  • the D1 sequence is designed to be different for each target, and SD and ED are designed to be common to all tag nucleic acids. Since these artificial arrays can be designed arbitrarily, it is possible to set a desired Tm value. Therefore, it is possible to carry out a reaction that is stable and has little mishybridization. For example, it is preferable to use an orthonormalized array as the artificial array.
  • An orthonormalized sequence is a sequence of nucleic acid molecules having a uniform Tm value, that is, a sequence designed so that the Tm values are aligned within a certain range, and the nucleic acid molecule itself is within the molecule.
  • (Intramolecular) is a sequence that does not inhibit hybridization with a complementary sequence, and a base sequence that does not form a stable hybrid other than a complementary base sequence. means. That is, a sequence included in one orthonormalized sequence group hardly reacts between sequences other than the desired combination and within a self-sequence, or does not generate a reaction.
  • the orthonormalized sequence when the orthonormalized sequence is amplified by PCR, the amount corresponding to the initial amount of the nucleic acid molecule having the orthonormalized sequence is not affected by the problems such as the above-mentioned crossnodification and hybridization.
  • This nucleic acid molecule has the property of being amplified quantitatively.
  • the above orthonormalized sequence is described in detail in H. Yshida and A Suyama, "Solution to 3—SAT by breadth first search", DIMACS Vol.54 9-20 (2 000) and Japanese Patent Application 2003-108126. Is described. Orthonormalized sequences can be designed using the methods described in these references.
  • a plurality of base sequences are randomly generated in advance, an average value of their melting temperatures is obtained, and candidate sequences are obtained based on a threshold value limited by ⁇ t ° C of the average value.
  • a method for preparing an orthonormalized sequence group from a candidate sequence obtained using whether or not the sequence reacts independently as an index is randomly generated in advance, an average value of their melting temperatures is obtained, and candidate sequences are obtained based on a threshold value limited by ⁇ t ° C of the average value.
  • a primer 1 having the same sequence as the SD sequence and a primer having a sequence complementary to the ED sequence labeled at the 5 'end are required.
  • a sample for detecting or quantifying the presence of the target nucleic acid, and a tag nucleic acid And the common probe are mixed, and the target nucleic acid is hybridized with the query probe portion of the tag nucleic acid and the common probe.
  • the second step of the present method is a step of linking a query probe portion of a tag nucleic acid hybridized to each corresponding target nucleic acid and a common probe.
  • the query probe portion and the common probe bind to the corresponding target nucleic acid, so that the ligation reaction is performed by ligase or the like, so that the query probe portion and the common probe are connected via the linking portion. Can be connected.
  • the linked tag nucleic acid and common probe are recovered.
  • the linked oligonucleotide can be extracted via the piotin label of the common probe by using magnetic beads with streptavidin bound to the surface.
  • the linked tag nucleic acid and common probe are dissociated from the corresponding target nucleic acid.
  • the target nucleic acid is present in the first solution, an oligonucleotide containing the corresponding Dl_sequence is extracted.
  • an amplification reaction is performed using an amplification method that yields a single-stranded amplification product, with the tag portion of the dissociated linking oligonucleotide as a template.
  • the amplified tag portion is detected.
  • typing according to the method of the present invention was performed according to the following procedure.
  • Encoding reaction including common probe, tag nucleic acid, and target nucleic acid are mixed and made into a hybrid, producing a linked oligonucleotide of tag nucleic acid and common probe, and recovering the linked oligonucleotide )
  • a region containing the target SNP was amplified from 5 ng of genomic DNA by multiplex PCR. This operation was performed according to the following procedure.
  • the thermal cycle for the reaction is as follows.
  • the thermal cycler was a Bio-Rad PTC-200.
  • the encoding reaction includes mixing and hybridizing the common probe, the tag nucleic acid, and the target nucleic acid, producing a linked oligonucleotide of the tag nucleic acid and the common probe, and recovering the linked oligonucleotide.
  • the reaction of 50 master mixes is as follows. Since New England Biolab Taq ligase was used for the ligase, the attached 10 X buffer was used. Tables 5 and 6 show the sequences of the common probe and tag nucleic acid.
  • Each tag sequence has a length of 23 bases.
  • the ply sequence 1 (SD), the sequence for identifying each allele (Dl_.), And the ply sequence 2 (ED) were arranged in the 5 'end.
  • Tag nucleic acid sequence Is an orthonormalized array.
  • the first genome PCR product is double-stranded, so it is not suitable for ligase reaction. Therefore, degeneration is performed.
  • the PCR reaction solution was stored frozen and once the enzyme activity was lost, the solution was denatured by the following procedure. First, it was denatured by heating at 95 ° C for 5 minutes. Immediately after denaturation, it was placed in ice, and the denatured DNA strands took an intramolecular structure, making them difficult to reassociate.
  • an encoding reaction solution having the following composition was prepared.
  • the Taq ligase used is from New England Biolab.
  • Ultrapure water was prepared by Milli-Q Synthesis from Millipore.
  • the encode reaction solution was reacted at the following temperature.
  • the solution composition is as follows.
  • the magnetic beads coated with streptavidin were Dynal ⁇ -280 magnetic beads.
  • a solution having the following composition was used as a B & W buffer according to the bead's instructions. [0076] Tris-HCl (pH 7.5) 10 mM
  • Streptavidin magnetic beads (hereinafter referred to as magnetic beads) are obtained by taking 1 ⁇ 1 from the stock solution containing preservatives and replacing the stock solution with B & W to 1 ⁇ 1 again. The solution thus prepared is shaken at room temperature for 15 minutes so that the magnetic beads are well dispersed in the solution.
  • This washing operation yielded magnetic beads from which non-specifically attached DNA was removed. This is called an encoded magnetic bead.
  • Primer 1 is a sequence corresponding to SD
  • primer 2 is a sequence corresponding to ED
  • Cy5-rED has a sequence corresponding to the complementary strand of ED and has a fluorescent dye Cy5 at the 5 'end. Is a label.
  • the ratio of SD: Cy5-rED is 1: 5, but it may be 1:10.
  • the concentration ratio may be appropriately selected if it is obtained in the state of a fluorescently labeled chain force i-strand as guasymmetric PCR. Since Takara Bio's Ex Taq was used as the polymerase, the 10-fold concentration buffer and dNTP mixture used for the preparation were the ones included in the kit. A 10-fold concentration buffer containing 20 mM magnesium ions was used.
  • the primer was diluted with ultrapure water. Here, if cycle elongation is performed, the primer amount should be set to 0 without one detection label.
  • the reaction solution was mixed with the encoded magnetic beads to disperse the beads.
  • the thermal cycle of the reaction is as follows.
  • the PTC-200 was used for the thermal cycler. If the cycle elongation method is used, the number of cycles is preferably 30 to 40 cycles.
  • the capillary array is a device that detects nucleic acids using a hybridization similar to a DNA microarray, and is probed with a probe force S along a groove-shaped channel.
  • the capillary array has 10 tag detection probes fixed in one groove, and the groove capacity is 25 1.
  • the slide is formed on a silicon rubber plate, and is attached to a slide glass in which the probe is spotted in a straight line using the adhesiveness of silicon rubber.
  • Silicone rubber is pasted in advance to fit the spot on the glass slide. Warm to C ⁇ .
  • the labeled PCR product was hybridized to this capillary array. Since magnetic beads remain in the asymmetric PCR solution, V was collected, and the magnetic beads were collected and collected as a supernatant.
  • This hybridization solution was warmed to 50 ° C. in advance, and 25 ⁇ l was injected into the similarly heated capillary array and hybridized at 50 ° C. for 30 minutes. A paper towel moistened with ultrapure water was laid so that the hybridization solution did not evaporate, and the hybridization was carried out by placing it in a tape cloth. Subsequently, washing was performed according to the following procedure.
  • Example 3 In order to confirm the further effect of the present invention, the inventors conducted a 96SNP co-typing experiment using the multiplex PCR method of the present invention! /, V, and an effective multiplex of shoes. It was verified whether amplification was performed. Since the success or failure of SNP typing is affected by the quality of the probe sequence of the SNP typing reaction, a typing failure does not necessarily indicate a failure of the multiplex amplification. In other words, SNPs that could be typed were because both amplification and probe detection were successful, and those that could not be typed were either amplification or probe detection either failed, or both were not successful. it is conceivable that.
  • SNPs that were sequenced using the Sangha method to obtain SNP sequences for controls were difficult to sequence and were not considered for PCR success. Also, depending on the sample population due to SNP, the probability of mutation is very low, and when alleles are present, even if a scatter diagram for type determination is drawn, cluster power may not appear. Such SNPs were excluded because their accuracy could not be evaluated.
  • the composition of the reaction solution is Titanium Taq Buffer (Takara BioClontech) 2 ⁇ 1
  • Primer mix (each primer 1 ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ) 0.74 ⁇ 1
  • Genomic DNA (5ng / 1) 2 ⁇ ⁇
  • the thermal cycle was as follows, and PTC-200 manufactured by Bio-Rad was used for the thermal cycler.
  • the encoding reaction conditions are slightly different from those of 10SNP.
  • the query uses ED and ED 'sequences depending on the alleles, so that they are labeled with different dyes at the detection stage so that each allele is detected with a different color fluorescence intensity on the same spot in the microarray.
  • Table 11 shows the common probe sequences and Table 12 shows the query probe sequences.
  • Table 12A Table 12 Query probe for 96 SNP
  • a liquid having the following composition was prepared.
  • a liquid having the following composition was prepared.
  • the hybridization solution should have a final concentration of So.5 X SSC, 0.1% SDS, 15% formamide, and lm M EDTA.
  • the solution obtained in the amplification reaction was combined with 101 to prepare 201, and hybridized with a hybrid array at 37 ° C for 60 minutes.
  • the silicon rubber groove was removed from the slide glass and washed with 0.1 SSC, 0.1% SDS washing solution for 5 minutes with shaking. Thereafter, the surface was quickly washed with ultrapure water, and the slide glass was dried and detected with GenePix 4000 of Molecular Devices.
  • QV Quality Value
  • This QV value was calculated for all the measurement points that make up the entire scatter plot, and the average value was taken as the QV value of the scatter plot (Equation 2).
  • the QV value is larger as the cluster to which the measurement point belongs is denser, the farther away the other cluster is closer, the larger the value, so the larger the QV value is, the better the scatter plot is, the better the SNP. It is thought that detection has been performed.
  • the scatter plot has a QV value of 44.74, and the vertical axis represents the fluorescence intensity value of the microarray.
  • the scatter diagram in Fig. 11 shows the detection result for SN P65, and the QV value was 44.74.
  • Table 15 shows the typing results of 96SNP.
  • the multiplex PCR method of the present invention accurately amplifies the target sequence with the SNP that was determined, and in the amplification of 66 SNP excluding 30 SNP from 96 SNP, PCR was performed with at least 53 of the 66 primers. It was a success. Some of the 13SNPs with poor scatter plot separation and inaccurate correct answers could be presumed to be successful in multiplex PCR, and the amplification success rate was considered to be even higher.

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Abstract

Procédé d'amplification PCR qui est du type amplification PCR multiplex caractérisé en ce que l'on utilise une amorce de 32 bases ou plus en longueur avant de conduire un recuit à une température inférieure à la valeur Tm de cette amorce et d'effectuer une réaction d'extension sur une longue période allant de 1 minute à 10 minutes.
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