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PCR (polymerase chain reaction) specificity improving method

A chain reaction and high specificity technology, which is applied in biochemical equipment and methods, microbial measurement/inspection, DNA preparation, etc., can solve the problem of little difference in binding stability, weak binding force, and inability to effectively improve the specificity of PCR reactions. sexual issues

Active Publication Date: 2016-04-13
CANCER CENT OF GUANGZHOU MEDICAL UNIV
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when the specificity of the target DNA sequence to be amplified is not high and there are many similar sequences in the template DNA, the 3' end of the primer may have multiple binding sites on the template DNA. In this case, PCR There will be non-specific amplification. When the non-specific amplification phenomenon is serious, it may even cause the target fragment to fail to amplify. At this time, the PCR reaction fails.
[0004] At present, the commonly used methods to improve the specificity of PCR reactions mainly include: (1) designing multiple primers to screen high-specificity primers, but when the specificity of the target sequence is poor, the effect of this scheme is not good; (2) improving the PCR reaction Annealing temperature: when the primer has multiple non-specific binding sites on the template, compared with the specific binding sites, most of the non-specific binding sites are only part of the complementary binding between the primer and the template, and its binding force is weak, Poor stability, so when the annealing temperature increases, the non-specific binding sites of the primers decrease, and the specificity of the PCR reaction increases; however, this method also has limitations. When the target sequence or primer specificity is poor, multiple The difference in the binding stability of the primer binding site is often not large, and increasing the annealing temperature cannot effectively improve the specificity of the PCR reaction; (3) Apply high-fidelity Taq enzyme: This method is to further improve the specificity of the PCR reaction when the specificity of the primer is acceptable. Reaction-specific method, so there are many non-specific amplifications that cannot be solved by this method

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  • PCR (polymerase chain reaction) specificity improving method
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  • PCR (polymerase chain reaction) specificity improving method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Example 1 PCR detection of epidermal growth factor receptor (EGFR) 18, 19, 20, 21 exons——20bp tail

[0103] In this example, exons 18, 19, 20, and 21 of the EGFR gene were selected as target sequences for testing and analysis. The reaction conditions of the conventional three-step method were adopted.

[0104] 1. Design of specific primers

[0105] Design specific PCR primers for exons 18-21 of the EGFR gene, 18F / 18R (amplified fragment length 271bp), 19F / 19R (amplified fragment length 304bp), 20F / 20R (amplified fragment length 242bp), 21F / 21R (the length of the amplified fragment is 262bp), the specific primer sequence is as follows (as shown in Table 1):

[0106] 18F (shown in SEQ ID NO.1): 5'-GGCTGAGGTGACCCTT-3';

[0107] 18R (shown in SEQ ID NO.2): 5'-GAGTAAAGTAGATGATGGAAATA-3'.

[0108] 19F (shown in SEQ ID NO.3): 5'-AGCATGTGGCACCATC-3';

[0109] 19R (shown in SEQ ID NO.4): 5'-CTAGAGCTAGAAAGGGAAAG-3'.

[0110] 20F (shown in SEQ ID NO.5): 5'-CCATGCGAAGCCCACA-3'...

Embodiment 2

[0133] Example 2 PCR detection of epidermal growth factor receptor (EGFR) 18, 19, 20, 21 exons——20bp tail

[0134] 1. Primers, tailing primers, and sample genomic DNA extraction are the same as in Example 1.

[0135] 2. Conventional three-step PCR detection

[0136] (1) PCR reaction system: 10 μL of TaqGreenmix, 1 μL of each upstream primer and downstream primer (primer concentration: 40 μmoL / L, 30 μmoL / L, 20 μmoL / L, 10 μmoL / L, 5 μmoL / L, 2.5 μmoL / L, 1.25 μmoL / L, 0.625 μmoL / L, 0.3125 μmoL / L), 6 μL of pure water, 2 μL of sample DNA, and a total volume of 20 μL.

[0137] PCR reaction conditions: 94°C for 3min; 40 cycles of 94°C for 30s, 56°C for 30s, 72°C for 45s; 72°C for 7min.

[0138] PCR product identification: PCR amplification products were identified by 2% agarose electrophoresis.

[0139] (2) The result is as follows figure 2 As shown, after the 5' end of the specific PCR detection primer was tailed, the detection specificity was significantly improved.

[0140] at...

Embodiment 3

[0142] Example 3 PCR detection of epidermal growth factor receptor (EGFR) exon 18, 19, 20, 21——15bp tailing

[0143] 1. The specific PCR primers 18F / 18R, 19F / 19R, 20F / 20R and 21F / 21R of the 18~21 exons of the EGFR gene designed in embodiment 1, and 5' tailed specific PCR primers (tail length 20bp), respectively designed and synthesized 5' tailed specific PCR primers with a 5' tailed length of 15 bp. The specific primer sequences after tailed are as follows:

[0144] 18F-15T (shown in SEQ ID NO.17): 5'-TGTTACTCGGGATTAGGCTGAGGTGACCCTT-3';

[0145] 18R-15T (shown in SEQ ID NO.18): 5'-CACGACTGAGAGCACGAGTAAAGTAGATGATGGAAATA-3'.

[0146] 19F-15T (shown in SEQ ID NO.19): 5'-GGTCAGTCGGCATTAAGCATGTGGCACCATC-3';

[0147] 19R-15T (shown in SEQ ID NO. 20): 5'-CACGACGGTCAGCACCCTAGAGCTAGAAAGGGAAAG-3'.

[0148] 20F-15T (shown in SEQ ID NO.21): 5'-AGTCAAGCGGAATTACCATGCGAAGCCCACA-3';

[0149] 20R-15T (shown in SEQ ID NO. 22): 5'-CACCACTCTCAGCACTCCCTTCCCCTGATTACCT-3'.

[0150] 21F-15T (sho...

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Abstract

The invention discloses a PCR (polymerase chain reaction) specificity improving method. In the method, a sequence which is nonhomologous from a template and incapable of forming stable secondary structure is added to the end 5' of a primer pair; further, reaction specificity is improved by adding tail primer to the end 5' on the basis of compound PCR conditions. The compound PCR conditions include, (1) 5-10 cycles of normal PCR reaction condition and (2) two-step PCR condition based on combination of high-temperature annealing and extension. Compared with normal PCR primer, the tailed primer can improve PCR specificity effectively without influence to flexibility; meanwhile, working concentration of the tailed primer is equal to or even lower than that of common primer, thus, PCR specificity can be improved remarkably on the normal PCR condition, and can be further improved by a way of combining with the compound PCR conditions. The method is adaptable to various PCR such as normal PCR and nested PCR, and is of high application value.

Description

technical field [0001] The invention belongs to the technical field of molecular biology. More specifically, it relates to a method of increasing the specificity of the polymerase chain reaction. Background technique [0002] Polymerase chain reaction (PCR) is a method widely used in biotechnology to efficiently amplify fragments of deoxyribonucleic acid (DNA). The basic principle of conventional PCR is: according to the known target DNA sequence to be amplified, design a pair of facing specific primers that are completely complementary to the two single strands of the target DNA sequence, and then template DNA, primers, DNA polymerase, 4 kinds of deoxygenated Nucleotides (dNTPs) are put into the reaction buffer, and then the target DNA fragment is amplified through the process of high temperature denaturation, low temperature renaturation, and medium temperature extension. In the high-temperature denaturation stage, the double-stranded DNA of the template is denatured and...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/11C12Q1/68
Inventor 罗凯贺智敏王倩张志杰郑国沛
Owner CANCER CENT OF GUANGZHOU MEDICAL UNIV
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