High-specificity multiplex-PCR (polymerase chain reaction) detection method for base mutation

A technology with high specificity and detection method, which is applied in the field of multiplex PCR detection of high specificity base mutation, to achieve the effect of improving detection specificity and sensitivity, high efficiency, and improving tissue heterogeneity

Inactive Publication Date: 2018-06-01
TIANJIN MEDICAL LAB BGI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The present invention aims to solve the problems of multiple PCR non-specific band interference and primer dimer, improve detection specifici

Method used

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  • High-specificity multiplex-PCR (polymerase chain reaction) detection method for base mutation
  • High-specificity multiplex-PCR (polymerase chain reaction) detection method for base mutation
  • High-specificity multiplex-PCR (polymerase chain reaction) detection method for base mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] To verify the gene mutation shown by the NGS data of a cancer patient, design 5 pairs of primers (see Table 4), and mix all the primers in equal proportions to form a primer combination.

[0071] The primer information table in the embodiment 1 of table 4

[0072]

[0073]

[0074] Use 5 pairs of primer combinations to carry out multiplex PCR on the patient's plasma DNA and blood cell DNA respectively (for the detection results of PCR products, see figure 2 ), after which a high-throughput sequencing library was constructed, and the sequencing results showed that the patient's JAK2 and DNMT3A genes were mutated, and the gene frequencies were 0.9% and 0.7% respectively (see Table 5).

[0075] Table 5 The sequencing results of Example 1

[0076] Chr.

Embodiment 2

[0078] To verify the gene mutation shown by the NGS data of a cancer patient, 4 pairs of primer pairs were designed (see Table 6), and all primers were mixed in equal proportions to form a primer combination.

[0079] The primer information table in the table 6 embodiment 2

[0080]

[0081] Use 4 pairs of primer combinations to carry out multiplex PCR on plasma DNA and blood cell DNA respectively (for the detection results of PCR products, see image 3 ), then a high-throughput sequencing library was constructed, and the sequencing results showed that the patient's DNMT3A gene was mutated, with a gene frequency of 0.85% (see Table 7).

[0082] Table 7 The sequencing results of Example 2

[0083]

Embodiment 3

[0085] 6 pairs of primers (see Table 9) were designed for hot spot mutations (see Table 8) of lung cancer with drug guidance significance, and all primers were mixed in equal proportions to form a primer combination. Multiplex PCR was performed on preoperative plasma DNA, blood cell DNA, and cancer tissue DNA from a patient with stage II non-small cell lung cancer. The product detection results are shown in Figure 4 , Hiseq sequencing results showed that relative to blood cell DNA, EGFR 19del somatic mutations appeared in both cancer tissue and plasma of the patient, and the gene frequency detection values ​​were 1.2% and 0.8%, respectively.

[0086] Table 8 Hotspot mutations in lung cancer

[0087]

[0088]

[0089]

[0090] Primer information table in table 9 embodiment 3

[0091]

[0092]

[0093] As an improvement or replacement of the present invention, the 5' end of the modified primer can be designed to connect the complementary universal sequence of th...

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Abstract

The invention discloses a high-specificity multiplex-PCR (polymerase chain reaction) detection method for base mutation. The method comprises steps as follows: under the action of DNA polymerase without 3'-5' exonuclease activity and with pyrophosphorolysis activity, multiple pairs of modified primers are utilized for PCR amplification in the presence of pyrophosphate, dideoxy nucleotide is located at 3' ends of upstream and downstream primers of the modified primers, the 3' ends are complementary with upstream and downstream sequences of a to-be-detected mutant type template, the DNA polymerase relies on the template to perform pyrophosphorolysis on dideoxy nucleotide located at the 3' ends of the modified primers and complementary with the template, the multiple pairs of primers are specifically bonded with the template and extend normally, and multiplex-PCR amplification is realized. According to the method, the problems of non-specificity strip interference and primer dimer of multiplex-PCR are solved, detection specificity and sensitivity are improved, and capacity of a system for the primers is increased; the method can be used for detecting body fluid DNA collected from cancer patients or healthy persons.

Description

technical field [0001] The invention relates to the technical field of gene mutation detection, in particular to a highly specific base mutation multiplex PCR detection method. Background technique [0002] Before the emergence of allele-specific PCR (allele specific PCR, AS-PCR) technology, the commonly used method for detecting gene mutations was to enrich the mutation point sequence by PCR and perform Sanger sequencing to detect multiple mutations. The quality of Sanger sequencing has requirements on the fragment length , greater than 100 bp and the mutation site is in the middle of the sequence, the detection result is more accurate, and the low quality of sequencing often occurs for small fragments below 100 bp; in addition, the sensitivity of Sanger sequencing can detect ≥ 5% of gene mutations, which obviously cannot meet the needs of short free DNA fragments Requirements for the detection of segmental low-frequency mutations. [0003] AS-PCR uses the principle of bas...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858
CPCC12Q1/6844C12Q1/6858C12Q2537/143C12Q2565/301C12Q2525/113
Inventor 刘磊宋炎张乐橦刘军朱红梅叶明芝
Owner TIANJIN MEDICAL LAB BGI
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