PCR-SSCP technology experiment

Summary

Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) technology can be used for: (1) screening and detection of oncogenes and oncogenic mutations; (2) analysis of causative genes of genetic diseases; (3) genetic diagnosis, gene mapping and other fields. diagnosis, gene mapping and other fields.

Operation method

PCR-SSCP technology experiment

Principle

The basic principle of PCR-SSCP technology is that PCR-amplified DNA fragments are denatured into single-stranded DNA, and single-stranded DNA forms different stereo conformations during electrophoresis in neutral polyacrylamide gel, and its conformation directly affects the rate of swimming, and the same length of single-stranded DNA with only a single difference in the order of the nucleotides of a single base can produce different stereo conformations, which results in the different rate of swimming and different bands of swimming. PCR amplified DNA fragments denatured into single-stranded after electrophoresis in the neutral gel without denaturant, and the normal comparison with the emergence of swimming band displacement can be presumed to exist base substitution. The nature of the base substitution must be determined by DNA sequencing. Therefore, PCR-SSCP is a common tool for mutation screening prior to sequencing.

Materials and Instruments

Sample DNA
Taq DNA polymerase PCR reaction buffer Primers dNTPs Acrylamide TBE buffer Ammonium persulfate TEMED Formamide sample buffer Fixation solution Silver staining solution Color development solution
Electrophoresis apparatus Water bath Electrophoresis tank

Move

I. PCR reaction

20 ml reaction system components are as follows:

template DNA (100 ng/ml) 1

ml10 × PCR reaction buffer 2

mlTaq DNA polymerase (5 U/ml) 0.2

ml4 × dNTPs (2.5 mmol/L) 1

mlMTHFR upstream and downstream primers 1

mlddH2O

each

to the end of the volume 20 ml

.

PCR reaction cycle parameters:

95 ℃ for 5 min;

94 ℃ for 30 s, 62 ℃ for 50 s, 72 ℃ for 90 s, a total of 30 cycles;

72 ℃ for 7 min.

After the end of the reaction, stored at 4 ℃.

II. Non-denaturing polyacrylamide gel electrophoresis

1. Preparation of 6% non-denaturing polyacrylamide gel: 30% polyacrylamide (crosslinking degree 49:1) 10 ml, 5 × TBE buffer 10 ml, add distilled water to 50 ml, add 25 ml of TEMED, 10% ammonium persulfate 250 ml, mixing and gel filling, inserting the sampling comb. After the gel is completely polymerized, pull out the spiking comb, install the electrophoresis device, add electrophoresis buffer (1×TBE), the buffer should be higher than the upper edge of the spiking hole, and flush the spiking hole with a syringe to suck up the buffer.

2. Take 5 ml of amplification product and add 10 ml of denaturing buffer to mix well, denature at 98℃ for 10 min, and ice bath quickly.

3. Take 5 ml of the mixture and add it to the sampling well, and electrophoresis with 1--8 V/cm for 4--5 h.

Polyacrylamide gel staining

1. Remove the electrophoresis device, take out the polyacrylamide gel, put it into a plastic disk, and rinse it with distilled water 1--2 times.

2. Pour in the fixing solution and fix it for 8-10 min. 3.

3. Rinse 1--2 times with distilled water; pour in the silver staining solution, silver staining 10--12 min. 4.

4. Rinse with distilled water for several times; pour in the color development solution and develop the color until a clear silver stained band appears. 5.

5. Soak the polyacrylamide gel in distilled water and observe the results of PCR-SSCP.

Judgment of results

Observe and record the DNA single-stranded bands on the polyacrylamide gel, and screen the mutant samples according to the abnormal swim shift.

Caveat

1. The length of the target DNA sequence should not be too long, otherwise the sensitivity will be reduced.

2. The length of the DNA sample swimming in the electric field is generally required to be more than 16--18 cm.

3. Staining should preferably be performed in a plastic dish.

4. The higher the ambient temperature, the easier the polyacrylamide gel solidifies. It should be adjusted according to the change of ambient temperatureThe amount of 10% ammonium persulfate and TEMED in the gel should be adjusted according to the change of ambient temperature.

5. maintain a constant temperature during electrophoresis, preferably using a thin gel and a cooling system to dissipate heat.

6. UseAgNO3When staining with AgNO 3, the staining time should be adjusted according to the ambient temperature, the lower the ambient temperature is, the more staining time is required.The lower the ambient temperature, the longer the dyeing time.

7. The color development time should not be too long, so as not to affect the observation of the results of the background is too dark.

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