PCR-SSP analysis experiment

Summary

PCR sequence-specific primer analysis can be used to: (1) detect sequence polymorphisms by PCR amplification of primers that specifically recognize a particular allele. (2) Detection of MN genotype.

Operation method

PCR-SSP analysis experiment

Principle

Based on the nature of the bases that determine a particular allele, sequence-specific primers are designed in which the first base at the 3′ end of the primer matches the specific base of each allele. During the PCR reaction, complete replication of the DNA fragments is achieved only if the first base at the 3′ end of the primer is complementary to the base that determines the particular allele, and the alleles are typed based on the presence or absence of the PCR product.

Materials and Instruments

Template DNA
Taq Polymerase Acrylamide Electrode Buffer Sampling Buffer
PCR Amplifiers Electrophoresis Instruments Electrophoresis Tanks

Move

1.PCR amplification PCR reaction system was 20 μl (2 systems, respectively, primer 1 and primer 2), containing 2 μl of template (about 50 ng), primers 1.5 μl each, dNTP 1.6 μl (0.4 mM), 10×Buffer buffer 2 μl, Taq enzyme 1.0 U, add double-distilled water to 20.0 μl; PCR reaction conditions were 94 ℃ 4min, 1.5min/52℃ 2.0min/72℃ 2.0min, 30 cycles. 94℃1.5min/52℃2.0min/72℃2.0min, 30 cycles.

2. Detection of PCR amplification products 2μl of PCR amplification products were taken and electrophoresed on 6% polyacrylamide gel (T=6%, C=3.3%, gel size 82mm×64mm×0.75mm).

The

electrode buffer is 1×TBE, and the digested product with 1/5 volume of sampling buffer is electrophoresed at 220 v for 2-3 h. Silver staining is used to develop the color

. ① If there is a base variant in the binding sequence of the primer, there may not be any amplification product, which may lead to wrong typing; ② The result can only be determined on the basis of the accuracy of typing of the control samples.

Caveat

1、If there is a base variant in the primer binding sequence, there may be no amplification product, resulting in incorrect typing.

2、The results can only be determined on the basis of accurate typing of control samples.

Common Problems

The method is simple to operate, and the primer design and complexity conditions of the experiment are demanding.

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