CN108103157B - High-specificity base mutation PCR detection method - Google Patents
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Abstract
The invention discloses a high-specificity base mutation PCR detection method, which comprises the following steps: firstly, performing nested PCR enrichment on template DNA; and then using DNA polymerase without 3'-5' exonuclease activity and with pyrophosphorolysis activity, carrying out PCR amplification by using a modified primer in the presence of pyrophosphate, wherein the corresponding position of the modified primer on the template is positioned on the inner side of the nested PCR primer, the 3 'end of the modified primer is dideoxynucleotide which is complementary to a mutant template to be detected but not complementary to a normal wild type template, the DNA polymerase depends on the template to pyrophosphorolysis the dideoxynucleotide of the 3' end of the modified primer, the primer extends normally, recording an amplification curve by using real-time fluorescence quantitative PCR, and detecting the Ct value difference. The method solves the problems of nonspecific band interference and primer dimer existing in the rapid detection of the low-frequency mutation of the base, and improves the detection specificity and sensitivity.
Description
Technical Field
The invention relates to the technical field of gene mutation detection, in particular to a high-specificity base mutation PCR detection method.
Background
Before the allele specific PCR (ALLELE SPECIFIC PCR, AS-PCR) technology appears, a commonly used method for detecting gene mutation is to enrich mutation point sequences by PCR to detect various mutations by Sanger sequencing, the quality of Sanger sequencing has requirements on fragment length, the mutation point is in the middle of the sequence, the detection result is more accurate, and the problem of low sequencing quality often appears for small fragments with the length of less than 100 bp; in addition, the sensitivity of Sanger sequencing can detect more than or equal to 5% of gene mutation, and obviously cannot meet the detection requirement of low-frequency mutation of free DNA short fragments.
AS-PCR utilizes the principle of base mismatch blocking amplification, designs a specific forward primer and a far-end reverse primer, wherein the 3' end of the primer is identical with the mutant genotype of SNP, only the mutant template can be amplified, and the wild template cannot be amplified. The specificity of the amplification reaction is increased, the mutation type template signal is amplified, and the sensitivity reaches 1%. The method can be used for detecting mutation with gene frequency less than or equal to 10%. The specificity was less than 2.5%.
In 2000, liu and Sommer were improved on the basis of AS-PCR, the 3 '-end deoxynucleotide dNMP of the primer was replaced with the dideoxynucleotide ddNMP, and only the 3' -end matched with the template was amplified using a DNA polymerase without 3'-5' exonuclease activity, which amplification was dependent on activation of pyrophosphorolysis, called pyrophosphorolysis polymerization (Pyrophosphorolysis-ACTIVATED POLYMERIZATION, abbreviated PAP). This improvement significantly increases the amplification specificity compared to conventional AS-PCR (Qin,J.,et al.,The Molecular Anatomy of Spontaneous Germline Mutations in Human Testes.PLoS Biology,2007.5(9):p.e224).
The prior art has the following disadvantages: (1) Non-specificity, because the PCR mismatch probability is 10 -7～10-5, the polymerase with the correction function can cut off the 3' end of the mismatch, and template-dependent amplification is carried out, so that a non-specific amplification product is generated, and false positive is caused; (2) Heterogeneity, the problem of heterogeneity generated by different sampling positions exists in that DNA (deoxyribonucleic acid) of a detection sample aiming at malignant tumor somatic mutation is collected from tumor tissues.
Disclosure of Invention
The invention aims to solve the problems of competitive amplification of wild type background and amplification inhibition of body fluid samples existing in rapid detection of low-frequency mutation of bases, and improve detection specificity and sensitivity; the kit can detect various sample DNA of tumor patients or healthy people, is a non-invasive sampling mode, and is more suitable for early screening of healthy people and post-operation drug detection.
Accordingly, the present invention provides a high-specificity base mutation PCR detection method comprising: firstly, performing nested PCR enrichment on template DNA; and then performing PCR amplification by using a DNA polymerase having no 3' -5' exonuclease activity and pyrophosphorolysis activity in the presence of pyrophosphate, using a modified primer, wherein the corresponding position of the modified primer on the template is positioned at the inner side of the nested PCR primer, the 3' end of the modified primer is a dideoxynucleotide, the 3' end of the modified primer is complementary to a mutant template to be detected and is not complementary to a normal wild template, the DNA polymerase pyrophosphorolysis of the dideoxynucleotide, the 3' end of the modified primer is complementary to the template, in the presence of the mutant template, the primer is normally extended, only the mutant template is amplified, and the amplification curve is recorded by using real-time fluorescent quantitative PCR to detect the difference of Ct values.
Further, the modified primer is a primer obtained by adding a dideoxynucleotide to the 3 '-end of the oligonucleotide in the presence of a terminal deoxynucleotidyl transferase and ddNTP, wherein the 3' -end of the oligonucleotide lacks the dideoxynucleotide.
Further, the template DNA is genomic DNA or episomal DNA.
Further, the above base mutation is a substitution, insertion or deletion.
Further, the above method is used for gene detection of various body fluid samples.
Further, the plurality of body fluid samples include plasma, cerebrospinal fluid, pleural effusion, saliva, ascites, exfoliated cells and lymph fluid.
Further, the above method is used for gene detection of tissue samples.
Further, the above method is used for gene detection of malignant tumor-associated somatic mutation.
Further, the 5' -end of the modified primer is ligated to a common sequence, and the PCR amplification is followed by a large amount of amplification using the common sequence.
Further, a tag sequence for distinguishing different samples is inserted into the common sequence, and a mixed library of PCR amplification products from different samples is subjected to high-throughput sequencing after the PCR amplification.
The method solves the problems of nonspecific band interference and primer dimer existing in the rapid detection of the low-frequency mutation of the base, improves the detection specificity and sensitivity to 0.1% -1%, and solves the problem of false positive of ASA-PCR. The method can detect various sample DNA (including blood plasma, cancer tissue, cerebrospinal fluid, pleural effusion, saliva, ascites, exfoliated cells, lymph fluid and the like) of a tumor patient or a healthy person, is a non-invasive sampling mode, and is more suitable for early screening of healthy people and postoperative drug detection. In addition, the humoral ctDNA can more comprehensively reflect malignant mutation than tissues, so that the problem of tissue heterogeneity can be improved to a certain extent, and the application range of gene detection is enlarged.
Drawings
FIG. 1 is a schematic diagram showing the reaction principle of the high-specificity base mutation PCR detection method of the present invention;
FIG. 2 is a mass spectrum of the deoxynucleotide chain before and after modification in example 1; wherein, the upper graph shows the deoxynucleotide chain before modification, the lower graph shows the modified deoxynucleotide chain, the abscissa shows the molecular weight, the molecular weight of the chl 9:5073770 forward primer is increased from 9000 to 9300 before modification, the increase is 300, and the average molecular weight of one dideoxynucleotide is about;
FIG. 3 is mass spectra of the deoxynucleotide chains before and after modification in examples 2 and 3; wherein, the upper graph shows the deoxynucleotide chain before modification, the lower graph shows the modified deoxynucleotide chain, the abscissa shows the molecular weight, the molecular weight of the chl 17:7578242 forward primer is increased from 5971 to 6271 before modification, the increase is 300, and the average molecular weight of one dideoxynucleotide is about;
FIG. 4 is a graph of the experimental results of example 1, wherein the top right hand corner graph shows a qPCR amplification curve, the plasma DNA nest product has significant amplification at the site, ct=12.11, and the blood cell DNA nest product has no amplification at the site; the upper left panel shows agarose electrophoresis, with plasma DNA (Sample) striped, and Wild-type (Wild) striped-free; the lower panel shows Sanger sequencing results, amplified using reverse primer, and found that the reverse strand product was mutated from the C at this position of the hg19 genomic sequence to A, indicating that the forward strand had a G.fwdarw.T mutation;
FIG. 5 is a graph of the experimental results of example 2, wherein the top right hand corner graph shows a qPCR amplification curve, the site of the pleural effusion DNA nest product is significantly amplified, ct=22.62, and the site of the blood cell DNA nest product is not amplified; the upper left panel shows agarose electrophoresis, with plasma DNA (Sample) striped, and Wild-type (Wild) striped-free; the lower panel shows Sanger sequencing results, amplified using reverse primer, and found that the reverse strand product was mutated from G at this position of the hg19 genomic sequence to A, indicating that the forward strand had a C.fwdarw.T mutation;
FIG. 6 is a graph of the experimental results of example 3, wherein the top right hand corner graph shows a qPCR amplification curve, the plasma DNA nest product has obvious amplification at the site, ct=20.77, and the blood cell DNA nest product has no amplification at the site; the upper left panel shows agarose electrophoresis, with plasma DNA (Sample) striped, and Wild-type (Wild) striped-free; the lower panel shows Sanger sequencing results, amplified using reverse primer, and found that the reverse strand product was mutated from G at this position of the hg19 genomic sequence to A, indicating that the forward strand had a C.fwdarw.T mutation.
Detailed Description
The invention will be described in further detail below with reference to the drawings by means of specific embodiments.
As shown in fig. 1, the principle of the method of the present invention is: firstly, performing nested PCR enrichment on genome DNA; then, using Taq DNA polymerase (Klentaq-S, SCIENTECH Corp.) with pyrophosphorolysis activity, which is free of 3' -5' exonuclease activity, DNA polymerase (e.g., bioengineered (F667Y and N-terminal deletion), in the presence of pyrophosphate, depending on the dideoxynucleotide of template pyrophosphorolysis primer whose 3' -end is complementary to the template, the primer is extended normally.
In a specific embodiment of the present invention, the highly specific base mutation PCR detection method of the present invention can be realized as follows.
1. Extraction of DNA
DNA is extracted from the sample.
2. Modified primers
A) The oligonucleotide ligation was carried out at 37℃for 4 hours as shown in Table 1.
TABLE 1
B) Urea denatured polyacrylamide gel electrophoresis (PAGE)
(1) Placing the solidified urea modified PAGE gel in a vertical electrophoresis tank containing 1 XTBE, and pre-electrophoresis for 30min under the condition of 250V constant pressure for standby;
(2) Taking out the sample from 37 ℃, putting the sample to room temperature, adding 15 mu l of bromophenol yellow loading buffer solution into each system (30 mu l), and uniformly mixing;
(3) Adding the sample into the bottom of a PAGE gel sample adding hole, carrying out constant-pressure electrophoresis at 200V for about 1h (the specific electrophoresis time depends on the length of the primer);
(4) After electrophoresis was completed, the PAGE gel was carefully removed and transferred to EB staining solution configured with 1XTBE and stained for 10min.
C) Recovery of fragments of interest
(1) Mashing the glue block, and adding a proper amount of 1 XNEB buffer2 (NEB) into the crushed glue block;
(2) Placing the suspension system into a 56 ℃ water bath kettle for water bath for 1h, and shaking and uniformly mixing every 15 min;
(3) Pouring the mixed system after water bath into a Spin-X centrifugal filter tube, and centrifuging at full speed for 1min; centrifuging, discarding the filter membrane and the broken rubber, and retaining the flow-through liquid in the collecting pipe;
(4) Measuring the volume of the fluid passing liquid by using a liquid transfer device; after measurement, 3 volumes of absolute ethanol, 20. Mu.l of 3M NaAc (pH 5.6) and 2. Mu.l of Glycogen (Glycogen) (5 mg/mL) were added to the flow-through liquid, mixed upside down and allowed to settle overnight at-20 ℃;
(5) Placing the alcohol precipitation system of the first day in a centrifuge, and centrifuging at a full speed of 4 ℃ for 30min;
(6) After centrifugation, the supernatant was carefully decanted, 700. Mu.l of 75% ethanol solution was added to the bottom pellet, and after mixing upside down, the pellet was centrifuged at 4℃for 10min at full speed;
(7) Repeating the steps for one time;
(8) The cover of the pipe is opened and is placed at a ventilation position for airing;
(9) Adding a proper amount of DNase-free water into the tube for precipitation and dissolving again;
(10) And detecting the change of the mass value by the time-of-flight mass spectrum, and determining whether the 3' -end of the primer is successfully modified.
3. Nested amplification
The sequence of the mutation site was amplified and enriched around the complementary sequence of the PAP primer, and the system is shown in Table 2.
TABLE 2
The procedure is as shown in table 3:
TABLE 3 Table 3
4. Purification
A) Taking out Ampure Beeds stored at 4deg.C, standing at room temperature for 30min, and balancing;
b) Adding a sample according to the volume of 1:1.5, fully and uniformly mixing, and standing at room temperature for 10min;
c) Placing the EP tube on a magnetic rack until the EP tube is clear;
d) Carefully aspirate the supernatant;
e) Adding 500 μl of 70% ethanol successively, blowing for 5-6 times, and discarding supernatant;
f) Repeating the previous step, and removing the supernatant as much as possible;
g) Placing metal Wen Yuyi ℃ and drying the magnetic beads;
h) Adding molecular biological water into the magnetic beads, fully and uniformly mixing the magnetic beads, standing for 10min, and then placing the magnetic beads in a magnetic rack until the magnetic beads are clarified;
i) Transferring the clarified liquid into a new EP pipe prepared in advance for storage;
j) Quantification of Qubit.
5. Real-time fluorescent Quantitative PCR (QPCR)
PCR and fluorescent signal capture were performed using a real-time fluorescent quantitative PCR instrument (e.g., stepone plus) standard curve method.
The real-time fluorescent quantitative PCR system is shown in Table 4.
TABLE 4 Table 4
The procedure is as shown in table 5:
TABLE 5
6. Agarose electrophoresis identification
A) 2% agarose gel 100V electrophoresis, using NEB 50bp Marker for comparison;
b) After electrophoresis, 1% ethidium bromide is used for dyeing glue, and image acquisition is carried out under an ultraviolet lamp;
c) Recovering a single target strip on a blue light adhesive cutting instrument;
d) Performing gel recovery by using a Qiagen gel recovery kit;
e) Qubit2.0 detection concentration;
f) ABI3730Sanger sequencing confirmed the point mutations.
The technical scheme and technical effects of the present invention are described in detail below through specific embodiments. The specific experimental procedures and methods in the following examples were performed as described above.
Example 1
Detecting whether G-T mutation occurs at 5073770 position of plasma DNA sample chr9:of a certain cancer patient. Primers were prepared with ddTMP ends (FIG. 2 shows that the addition of the modified primer ends was successful), nested PCR (primer sequences see Table 6) was performed on the sample DNA, the sequence of the mutation was enriched, 4ng was used for real-time fluorescent quantitative PCR detection (primer sequences see Table 6) after purification, electrophoresis was recovered from the product, sanger sequencing was performed on the target band recovery, QPCR resulted in significant amplification of the sample, but no amplification was performed on the wild-type template, and Sanger sequencing resulted in the mutation of the positive strand guanine nucleotide to thymine nucleotide at that position (see FIG. 4). The patient's plasma DNA high throughput sequencing showed a mutation frequency of 0.6%.
TABLE 6 primer information Table in example 1
Example 2
Whether the C-T mutation occurs at the 7578242 position of a pleural effusion cfDNA sample chr17 of a patient with non-small cell lung cancer is detected, and cannot be verified by a mass spectrum or a first-generation sequencing method. Primers were prepared with ddTMP ends (FIG. 3 shows that the addition of the modified primer ends was successful), nested PCR (primer sequences see Table 7) was performed on the sample DNA, the sequence of the mutation was enriched, 4ng was used for real-time fluorescent quantitative PCR detection (primer sequences see Table 7) after purification, electrophoresis was recovered from the product, sanger sequencing was performed on the target band recovery, QPCR resulted in significant amplification of the sample, but no amplification of the wild type template, and Sanger sequencing resulted in the mutation of the positive strand cytosine nucleotide to thymidine at this position (see FIG. 5). The patient's cancer tissue DNA high throughput sequencing data showed a mutation frequency of about 0.1%.
TABLE 7 primer information Table in example 2
Example 3
Detecting whether C-T mutation occurs at the 7578242 position of a patient plasma DNA sample chr17 in example 2, preparing a primer with a ddTMP end (FIG. 3 shows that the addition of the ddTMP at the end of the modified primer is successful), performing nested PCR (primer sequence is the same as in example 2) on cfDNA of the sample plasma, enriching the sequence of the mutation, taking 4ng for detection after purification (primer sequence is the same as in example 2), carrying out product recovery electrophoresis, carrying out Sanger sequencing on the target strip recovery, carrying out QPCR result, wherein the sample has obvious amplification, and a wild template has no amplification, and carrying out Sanger sequencing result shows that the mutation of positive strand cytidine acid at the position into thymidylic acid (see FIG. 6). The patient plasma DNA high throughput sequencing data showed a mutation frequency of 0.3%.
As other alternative or improvement modes of the invention, the PAP-PCR method of the invention can also be used for real-time fluorescence quantitative PCR detection by a probe method, and can also be combined with ddPCR to accurately quantify mutation frequency, and the requirements of a ddPCR platform on templates are also more relaxed. The 5' end of the primer can be designed to be connected with a public sequence, PAP-PCR is carried out after mass amplification, or a tag (index) sequence for distinguishing different samples is inserted into the public sequence, and PAP-PCR can be carried out in a mixed library (pooling) mode for high-throughput sequencing. In addition, the 3' end of the primer may use a locked nucleic acid instead of a dideoxynucleotide. Locked nucleic acid LNA (Locked Nucleic Acid) is an oligonucleotide-like derivative comprising six bases, A, C, G, T, U, and mC. The 2'-O and 4' -C positions of the beta-D-ribofuranose in the structure form a rigid structure through the action of shrinkage, so that the flexibility of the ribose structure is reduced, and the stability of the local structure of the phosphate skeleton is improved. Single LNA can raise the annealing temperature of heterogeneous nucleic acid double chain 1～8℃(Zeng,Y.,et al.,Establishment of Real Time Allele Specific Locked Nucleic Acid Quantitative PCR for Detection of HBV YIDD(ATT)Mutation and Evaluation of Its Application.PLoS One,2014.9(2):p.e90029)., and LNA and DNA/RNA have the same phosphate skeleton in structure, so that the LNA has good recognition capability and strong affinity to DNA and RNA. In the PCR annealing step, if the 3' -end LNA of the primer can be complementary with the template, the amplification product can be obtained by continuous extension, otherwise, the amplification product cannot be extended. Multiple PCR was performed on multiple pairs of PAP primers, see Ampliseq technology.
The foregoing is a further detailed description of the invention in connection with specific embodiments, and it is not intended that the invention be limited to such description. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the invention, and these should be considered to be within the scope of the invention.
SEQUENCE LISTING
<110> Tianjin Huada medical test all companies; shenzhen Hua big Gene stock Co., ltd; guangzhou Hua big Gene medicine inspection all Limited
<120> A high specificity base mutation PCR detection method
<130> 16I23168
<160> 8
<170> PatentIn version 3.3
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agcaagtatg atgagcaagc t 21
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gctgtgatcc tgaaactgaa ttt 23
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agcatttggt tttaaattat ggagtatgtt 30
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cagagacccc agttgcaaac 20
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Claims (6)
1. The application of the primer group in preparing a high-specificity base mutation PCR detection reagent is characterized in that the primer group is used for base mutation PCR detection based on the following method, and the method comprises the following steps: firstly, performing nested PCR enrichment on template DNA; then using DNA polymerase without 3'-5' exonuclease activity and pyrophosphorolysis activity to carry out PCR amplification in the presence of pyrophosphate, using a forward primer and a reverse primer, wherein the positions of the forward primer and the reverse primer corresponding to the template are positioned on the inner side of a nested PCR primer, the forward primer is a modified primer, the 3 'end of the modified primer is dideoxynucleotide, the 3' end of the modified primer is complementary to a mutant template to be detected and is not complementary to a normal wild type template, the 3 'end of the reverse primer is not dideoxynucleotide, the 3' ends of the forward primer and the reverse primer are not overlapped, the DNA polymerase is dependent on the template to pyrophosphorolysis of the dideoxynucleotide complementary to the template in the presence of the mutant template to be detected, the primer is normally extended, and the amplification curve is recorded by using real-time fluorescence quantitative PCR to detect the Ct value difference;
The template DNA is free DNA;
the mutation is a mutation of G-T at 5073770 position of chr9 or a mutation of C-T at 7578242 position of chr17;
For the mutation detection of the position G-T of chr9:5073770, in the nested PCR, the nested forward primer is SEQ ID NO:1, the nested reverse primer is SEQ ID NO: 2; in the PCR amplification using the forward primer and the reverse primer, the forward primer is AGCATTTGGTTTTAAATTATGGAGTATGTDDT, and the reverse primer is SEQ ID NO: 4;
For the mutation detection of the position G-T of chr17:7578242, in the nested PCR, the nested forward primer is SEQ ID NO:5, the nested reverse primer is SEQ ID NO: 6; in the PCR amplification using the forward primer and the reverse primer, the forward primer is TGTTTCTGTCATCCAAATACTCCADDT, and the reverse primer is SEQ ID NO: 8.
2. The use according to claim 1, wherein the modified primer is a primer obtained by adding a dideoxynucleotide to the 3 '-end of an oligonucleotide synthesized in advance in the presence of a terminal deoxynucleotidyl transferase and ddNTP, in the absence of the dideoxynucleotide at the 3' -end.
3. The use according to claim 1, wherein the template DNA is derived from a plurality of body fluid samples.
4. The use of claim 3, wherein the plurality of body fluid samples comprises plasma, cancerous tissue, cerebrospinal fluid, pleural effusion, saliva, ascites, shed cells, and lymph fluid.
5. The use of claim 1, wherein the 5' end of the modified primer is ligated to a common sequence, and the PCR amplification is followed by a large number of amplifications using the common sequence.
6. The use according to claim 5, wherein the common sequence is inserted with a tag sequence for distinguishing between different samples, and wherein the PCR amplification is followed by high throughput sequencing of a pool of PCR amplification products derived from different samples.
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| CN101381766A (en) * | 2008-06-11 | 2009-03-11 | 厦门大学 | Quantitative detection method of gene rare mutation |
| CN104450869A (en) * | 2013-09-12 | 2015-03-25 | 中国科学院上海巴斯德研究所 | Dideoxyribonucleoside modified primer, method, reaction system and application thereof to mutation detection |
| CN104789643A (en) * | 2014-01-21 | 2015-07-22 | 中国科学院上海巴斯德研究所 | Double deoxidation modified primer and kit used for detecting EGFR gene mutation, and applications of double deoxidation modified primer and kit |
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| CN101381766A (en) * | 2008-06-11 | 2009-03-11 | 厦门大学 | Quantitative detection method of gene rare mutation |
| CN104450869A (en) * | 2013-09-12 | 2015-03-25 | 中国科学院上海巴斯德研究所 | Dideoxyribonucleoside modified primer, method, reaction system and application thereof to mutation detection |
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