Allele-specific PCR

Summary

Allele-specific PCR, also known as mismatch PCR, amplification refractory mutation system (ARMS), and mismatch amplification mutation assay (MAMA), is an alternative method to single-strand conformation polymorphism (SSCP) and restriction fragment length polymorphism (RFLP). MAMA), which is a more efficient method than single-strand conformation polymorphism (SSCP), restriction fragment length polymorphism (RFLP), heteroduplexanalysis (HDPA), or heterologous double-strand analysis (HDA). heteroduplexanalysis (HA) are more simple, rapid and easy to detect the PCR method for a large number of specimens.

Principle

The basic principle of allele-specific PCR is that Taq DNA polymerase lacks exonuclease activity, and under certain conditions, the mismatch at the 3' end of the PCR primers leads to a drastic reduction of the product, and the design of appropriate primers for the different known mutations can achieve the purpose of distinguishing between mutant and wild-type genes directly by PCR. This method requires the preparation of three PCR primers, two of which contain the mutated bases and normal bases of the mutated site in the DNA to be tested, and the other one is the normal opposite primer. After PCR amplification, the normal primers amplify only the normal DNA, while the primers containing the mutated bases amplify the mutated DNA strand, and then the normal agarose gel electrophoresis is used to detect the presence or absence of amplification products.

According to the known mutated and conserved regions in the genome, three primers were designed, among which two upstream primers were designed, and the difference was only the base at the end of 3', one was allele 1-specific primer (ASP1, A at the end of 3'), and the other was allele 2-specific primer (ASP2, C at the end of 3'), and the downstream primer (CON) was designed in a relatively conserved position. Then, for the genomes containing allele 1 and allele 2, PCR was performed with ASP1, ASP2 and the downstream conserved primer (CON), respectively, under the same reaction conditions. The results were then analyzed. When allele 1 DNA was used as the template, PCR with ASP1 paired with a downstream conserved primer (CON) resulted in effective amplification, while PCR with ASP2 paired with a downstream conserved primer (CON) resulted in no amplification; when allele 2 DNA was used as the template, PCR with ASP2 paired with a downstream conserved primer (CON) resulted in effective amplification, while PCR with ASP1 paired with a downstream conserved primer (CON) resulted in no amplification. When allele 2DNA was used as the template, PCR with ASP2 paired with the downstream conserved primer (CON) resulted in effective amplification, while PCR with ASP1 paired with the downstream conserved primer (CON) resulted in no amplification.

Operation method

Allele-specific PCR

Principle

The basic principle of allele-specific PCR is that Taq DNA polymerase lacks exonuclease activity, and under certain conditions, the mismatch at the 3' end of the PCR primers leads to a drastic reduction of the product, and the design of appropriate primers for the different known mutations can achieve the purpose of distinguishing between mutant and wild-type genes directly by PCR. This method requires the preparation of three PCR primers, two of which contain the mutated bases and normal bases of the mutated site in the DNA to be tested, and the other one is the normal opposite primer. After PCR amplification, the normal primers amplify only the normal DNA, while the primers containing the mutated bases amplify the mutated DNA strand, and then the normal agarose gel electrophoresis is used to detect the presence or absence of amplification products. According to the known mutated and conserved regions in the genome, three primers were designed, among which two upstream primers were designed, and the difference was only the base at the end of 3', one was allele 1-specific primer (ASP1, A at the end of 3'), and the other was allele 2-specific primer (ASP2, C at the end of 3'), and the downstream primer (CON) was designed in a relatively conserved position. Then, for the genomes containing allele 1 and allele 2, PCR was performed with ASP1, ASP2 and the downstream conserved primer (CON), respectively, under the same reaction conditions. The results were then analyzed. When allele 1 DNA was used as the template, PCR with ASP1 paired with a downstream conserved primer (CON) resulted in effective amplification, while PCR with ASP2 paired with a downstream conserved primer (CON) resulted in no amplification; when allele 2 DNA was used as the template, PCR with ASP2 paired with a downstream conserved primer (CON) resulted in effective amplification, while PCR with ASP1 paired with a downstream conserved primer (CON) resulted in no amplification. When allele 2DNA was used as the template, PCR with ASP2 paired with the downstream conserved primer (CON) resulted in effective amplification, while PCR with ASP1 paired with the downstream conserved primer (CON) resulted in no amplification.

Materials and Instruments

Equipment: PCR amplifier.
Reagents:
① Template: genomic DNA or serum.
② dNTP mixture: the concentration of each deoxyribonucleic acid is 2.5 mmol/L. ③ Three specific primers: the concentration is 10 pimol/L.
③ Three specific primers: the concentration of each is 10 pimol/L. ④ Taq DNA polymerase.
④ Taq DNA polymerase.
⑤ 10 × PCR buffer: 15 mmol/L MgCl
⑤ 10 × PCR buffer: 15 mmol/L MgCl2
⑤ 10 × PCR buffer: 15 mmol/L MgCl2, 500 mmol/L KCl, 100 mmol/L Tris-Cl, 0.1% (v/v) Triton X-100.
(vi) Autoclaved deionized water.
(vii) Reagents for agarose gel electrophoresis.

Move

The basic process of allele-specific PCR can be divided into the following steps:

(i) Primer design

This is the most important part of allele-specific PCR. Based on the known allelic mutations, the 3' ends of the primers are designed to be at the positions where the mutations are likely to occur. One primer can be fully complementary to the allele 1 fragment (the 3' end does not match allele 2), the other primer can be fully complementary to the allele 2 fragment (the 3' end does not match allele 1), and the reverse primer can be designed to be in the more conserved region, so that the amplified fragment is usually within 300 bp (see Notes ②). （The reverse primer is designed to be within the more conserved region, and the amplified fragment is usually within 300 bp (see Note ②)

(ii) Template

Generally, extracted genomic DNA (about 50 ng) is used, and the use of blood samples has also been reported.

(iii) Procedure

1. Set up 50 μl of PCR reaction system, prepare at least two tubes of reaction solution in 0.25 ml PCR tubes, and add the following components separately.

Note: ASP1: Allele 1-specific primer; ASP2: Allele 2-specific primer: CON: downstream primer.

If the PCR instrument does not have a thermal cap, add a drop of mineral oil (about 50 μl) to the reaction solution. Place the PCR tube on the PCR instrument. 2.

2. Set up the PCR reaction conditions

3. Detection and analysis of PCR products: At the end of the reaction, 10 μl of the reaction solution is analyzed by agarose gel electrophoresis. If the test is successful, primers containing alleles 1 and 2 should amplify on their respective templates.

Alternatively, more sophisticated assays can be used. Wang et al. separated the PCR products by electrophoresis with the ABI Prism™ 310 Ge-netic Analyzer. 1 μl of the PCR product was spiked into 12 μl of formamide, and 0.5 μl of GeneScan™ Size Standards LIZ-500 (orange) was added as an internal standard and electrophoresed in capillary tubes for 25 min. (1.5 kV, POP4 gel, 47 cm capillary tube). At the end of the electrophoresis, the products were analyzed by GeneScan™ V3.0. SNP typing was performed according to the length of the product and the number of product peaks to determine the genotype of each SNP locus.

Caveat

In allele-specific PCR, how to control the appearance of false-positive products is a very critical issue. Usually, the first step is to start from the reaction system and conditions of PCR, and the methods adopted include lowering the amount of Taq enzyme, primer concentration, dNTP concentration, and increasing the annealing temperature of the PCR reaction, etc. The optimal conditions for each reaction should be determined through several different pre-tests. The optimal conditions for each reaction should be determined through several different pre-tests to determine the best combination.② When designing primers, it is important to note that the mismatch must be at the last base of the 3' primer, otherwise it will not be possible to judge whether the mutation has occurred or not. Occasionally, the mismatch at the 3' end of the primer is not enough to achieve the desired level of resolution, especially when the ratio of mutant to wild type is low. In this case, the resolution can be significantly improved by introducing an artificial mismatch at the penultimate or third base of the 3' end.In allele-specific PCR, the use of a single primer pair is usually prone to false-negative results. The introduction of multiplexed PCR and the design of a second primer pair or primer at the periphery of the PCR product to be amplified can serve as an internal positive control to avoid false-negative results.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/