Experimental principle
Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) technology is developed on the basis of PCR technology. It is a simple, fast and economical display. A means of single base mutation (point mutation) in the PCR reaction product. This method has been used as a screening test for oncogene and tumor suppressor gene mutations, pathogenic gene analysis and genetic diagnosis of genetic diseases, and gene mapping.
In the SSCP assay, double-stranded DNA (dsDNA) is denatured into single-stranded DNA (ssDNA), and each single-stranded DNA exhibits a unique folded conformation based on their internal sequences, even if the same length of DNA is single-stranded. Different conformations are formed due to differences in base sequences and even individual bases. These single-stranded DNAs were separated by non-denaturing polyacrylamide gel electrophoresis under non-denaturing conditions. The mobility and banding pattern of single-stranded DNA depend on their folded conformation and temperature during electrophoresis.
Reagents and materials
1. Sample DNA (100 ng/ml), MTHFR upstream and downstream primers (5 pmol/ml), Taq DNA polymerase (5 U/ml), 10×PCR reaction buffer, 4×dNTPs (2.5 mmol/L), 30% Acrylamide (degree of crosslinking 49:1), 5×TBE buffer, 10% ammonium persulfate (currently available), TEMED, formamide loading buffer, fixative, silver stain, and color developing solution.
2. Micro-sampler, PCR automatic thermal cycler, polyacrylamide gel electrophoresis device.
Experimental procedure
First, PCR reaction
The composition of the 20 ml reaction system is as follows:
Template DNA (100 ng/ml) 1 ml
10×PCR Reaction Buffer 2 ml
Taq DNA Polymerase (5 U/ml) 0.2 ml
4×dNTPs (2.5 mmol/L) 1 ml
MTHFR upstream and downstream primers 1 ml each
ddH2O is added to the final volume of 20 ml
PCR reaction cycle parameters:
95 ° C 5 min;
94 ° C 30 s, 62 ° C 50 s, 72 ° C 90 s, a total of 30 cycles;
72 ° C for 7 min.
After the reaction was completed, it was stored at 4 ° C.
Second, non-denaturing polyacrylamide gel electrophoresis
1. Preparation of 6% non-denaturing polyacrylamide gel: 30% polyacrylamide (degree of crosslinking 49:1) 10 ml, 5 × TBE buffer 10 ml, add distilled water to 50 ml, add TEMED 25 ml, 10% 250 ml of ammonium sulfate, mix and mix, and insert the sample comb. After the gel is completely polymerized, pull out the sample comb, install the electrophoresis device, add the electrophoresis buffer (1×TBE), the buffer should be higher than the upper edge of the sample well, and use a syringe to absorb the buffer and rinse the sample well.
2. Mix 5 ml of amplified product and add 10 ml of denatured loading buffer, denature at 98 °C for 10 min, and quickly ice bath.
3. 5 ml of the mixture was added to the wells and electrophoresed at 1 to 8 V/cm for 4 to 5 hours.
Third, polyacrylamide gel dyeing
1. Remove the electrophoresis device, remove the polyacrylamide gel, place it in a plastic tray, and rinse it with distilled water for 1-2 times.
2. Pour in the fixative and fix for 8 to 10 minutes.
3. Rinse with distilled water for 1 to 2 times; pour in silver stain solution and stain with silver for 10 to 12 minutes.
4. Rinse several times with distilled water; pour in the coloring solution and develop a clear silver stained strip.
5. The polyacrylamide gel was soaked in distilled water and the PCR-SSCP results were observed.
Result judgment
The DNA single-stranded band on the polyacrylamide gel was observed and recorded, and the mutant sample was screened according to the abnormal migration displacement.
application
There are a wide range of applications in genetics, such as screening for gene mutations in oncogenes or tumor suppressor genes, mutation screening for disease-causing genes in genetic diseases, and genetic diagnosis.
Precautions
1. The length of the target DNA sequence should not be too long, otherwise the sensitivity will decrease.
2. The length of the DNA sample swimming in the electric field is generally required to be 16 to 18 cm or more.
3. The dyeing is preferably carried out in a plastic tray.
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