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High-efficiency fluorescent allele-specific polymerase chain reaction method and detection genotyping method based on automatically designed primers

An allele-specific, chain reaction technology, applied in biochemical equipment and methods, microbial measurement/testing, DNA preparation, etc., can solve the problems of low reaction sensitivity and high site design failure rate, and achieve high detection The effect of specificity, cheap detection, and stable reaction

Active Publication Date: 2019-01-11
深兰国开基因科技(山东)有限公司
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Problems solved by technology

[0011] In order to overcome the above-mentioned deficiencies, the present invention aims to provide an automatic primer design method for allele-specific polymerase chain reaction, and the automatic primer design method for this allele-specific polymerase chain reaction The method is to solve the technical problems of low allele-specific polymerase chain reaction sensitivity and high site design failure rate in the prior art

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  • High-efficiency fluorescent allele-specific polymerase chain reaction method and detection genotyping method based on automatically designed primers
  • High-efficiency fluorescent allele-specific polymerase chain reaction method and detection genotyping method based on automatically designed primers
  • High-efficiency fluorescent allele-specific polymerase chain reaction method and detection genotyping method based on automatically designed primers

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Embodiment 1

[0031] Embodiment 1: as figure 1 As shown, for automatic design of a SNP site, the sequence of about 50 bp upstream and downstream of the site must be obtained first, and the design of any internal primer starts from the 3' end. The present invention is different from the traditional AS-PCR in that the 3' ends of both the upstream and downstream primers are designed to have specific matching on the SNP site (the matching site of the SNP is at -1 to -3 positions of the 3' end). Therefore, the positive and negative strand primers also overlap at the 3' end (1-5bp overlap). Then extend from the 3' end to the 5' end, and determine a sequence that matches the template (reverse complement) on the positive and negative strands. The annealing temperature of the primer (defined as the internal primer temperature in this paper) needs to be within the specified range (44-60 degrees, the ideal temperature is 51 degrees). Calculated using a molecular thermodynamic model of the primers. ...

Embodiment 2

[0042] Example 2: High-efficiency fluorescent allele-specific polymerase chain extension (UFAS-PCR) reaction using the primers designed in Example 1:

[0043] (1) The ratio of the reaction system is shown in the following table 2, taking the 3ul system suitable for a 384-well plate as an example:

[0044] double distilled water

0.568μl

10x buffer

0.3μl

dNTP

0.06μl

Mg 2+

0.06μl

0.6μm primer

1μl

Taq DNA polymerase

0.012μl

Template DNA (1.5ng / μl)

1μl

overall system

3μl

[0045] Table 2

[0046] (2) 384-well plate detection sample loading format is as follows: figure 2 As shown, the odd-numbered rows (A, C, E, G, I, K, M) plus the primers and reaction system of the first allele (marked by a1); the even-numbered rows (B, D, F, H, J, L, N) plus the primers of the second allele and the reaction system (marked by a2). Add the same sample to each pair of wells.

[0047] (3) PCR reactio...

Embodiment 3

[0050] Example 3: real-time fluorescence detection, 0.15ul of water in the system of Example 2 was replaced with evagreen or similar fluorescent dyes for real-time fluorescent PCR, and the fluorescence value in the system was detected during the low temperature period of each PCR cycle. Evagreen fluorescent dyes refer to fluorescent dyes that do not seriously interfere with the PCR reaction and can bind to DNA double strands independently of sequence specificity. The intensity of the fluorescent signal can reflect the total amount of double-stranded DNA in the system linearly or nonlinearly.

[0051] The method for real-time fluorescence detection can carry out more than 30 (optimum condition 60) PCR cycles according to the reaction conditions of Example 2, and do a real-time PCR reaction respectively for the two alleles, thereby reading the SNP from the real-time curve genotype. The exponential growth of the positive reaction curve occurs much earlier than the negative reacti...

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Abstract

The invention relates to an automatic primer design method for AS-PCR (allele-specific-polymerase chain reaction), and provides a method adopting primers designed with the automatic primer design method for efficient fluorescent AS-PCR as well as a gene typing detection method. For any SNP (single nucleotide polymorphism) locus, four inner primers are designed on the basis of sequences of 40-60 bp at upstream and downstream parts of the SNP locus, the design of any one of the inner primers is started from a 3' terminal, the 3'terminal of any one of the inner primers has specificity matching at the SNP locus, and matching loci of the SNP locus are at (-1)-(-3) positions of the 3' terminal of the primers; after the inner primers are obtained, a short segment which is not matched with template specificity is added to the 5' terminal of each inner primer, and the inner primers and the short segments jointly constitute outer primers. According to the invention, high specificity detection of gene typing analysis can be realized under the same reaction condition.

Description

technical field [0001] The invention relates to the field of biological genes, in particular to an allele-specific polymerase chain reaction (AS-PCR) reaction, specifically an efficient fluorescent allele-specific polymerase chain reaction based on automatically designed primers Methods and detection genotyping methods. Background technique [0002] With the in-depth study of the human genome, genetic testing is increasingly showing an important role. In recent years, most of the detections are aimed at single nucleotide diversity (SNP). Because it is the most ubiquitous in the genome (accounting for more than 95%), the simplest form (one base changes to another base), and the most well-studied (the position and frequency of millions of SNP sites are very clear, they There are a lot of researches on the relationship between it and diseases). SNP typing analysis not only has a wide demand in scientific research, but also with the extensive development of association studie...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6858C12N15/11C12N15/10
CPCC12Q1/6827C12Q1/6858C12Q2561/113C12Q2563/107C12Q2531/113
Inventor 秦鹏飞
Owner 深兰国开基因科技(山东)有限公司