PCR technology topic: Restriction fragment length polymorphism (RFLP) analysis
Restriction fragment length polymorphism (RFLP) analysis is one of the important analytical methods in molecular biology for the detection of DNA sequence polymorphisms. PCR-RFLP combines PCR technology, RFLP analysis and electrophoresis methods to replicate and amplify the target DNA fragment to be tested, then digest the amplified product with DNA restriction endonuclease, and finally analyze the target by electrophoresis. Whether the DNA fragment is cut and typed.
In this experiment, RFLP analysis was used to detect two SNP loci rs17750303 (A→C) and rs837690 (A→G) in the intron 3 of the GRIN2A subunit gene in the NMDA receptor.
[Experimental principle]
DNA restriction enzymes have an active function of recognizing a specific DNA sequence and cleavage of a DNA duplex at a specific site, and the DNA molecule is altered (or formed) by a mutation (nucleotide substitution, insertion or deletion). The enzyme recognition sequence prevents the DNA restriction endonuclease from (or can) cleave the target DNA fragment, and when the electrophoresis is detected, the original DNA fragment of the corresponding DNA restriction endonuclease recognition sequence becomes a short fragment; there is no corresponding DNA restriction The length of the original DNA fragment of the endonuclease recognition sequence will not change.
[experiment equipment]
PCR instrument, electrophoresis instrument, electrophoresis tank, microwave oven, incubator or constant temperature water bath.
[Experimental reagent]
Restriction enzyme BstNI, template DNA, Taq polymerase, PCR primer (upstream primer: 5-CTAAGCCTTCCCTTTATCACC-3'; downstream primer: 5-TTCACAGTCGTGCTTGTTTCT-3'), acrylamide, enzyme digestion buffer, electrode buffer, Load buffer, etc.
[experimental method]
1. PCR amplification: PCR reaction system is 20μl, containing template 2μl (about 50ng), primers 1.6μl, dNTP1.6μl (0.4mM), 10×Buffer buffer 2μl, Taq enzyme 0.5U, add double distilled water to 20.0 μl; PCR reaction conditions were 95 ° C 2 min, 94 ° C 30 sec / 64 ° C 30 sec / 72 ° C 30 sec (5 cycles), 94 ° C 30 sec / 63 ° C 30 sec / 72 ° C 30 sec (5 cycles), 94 ° C 30 sec / 60 °C30sec/72°C 25sec (23 cycles), 94°C 30sec/60°C 30sec/72°C for 1min.
2. Detection of PCR amplification products: 2 μl of the PCR amplification product was taken, and the amplification product of the target DNA fragment was detected by 1% agarose gel diving electrophoresis.
3. Enzyme digestion reaction: Take about 2.0 μl of the PCR product, add restriction endonuclease BstN I1U, 1.0×1× digestion reaction buffer 1.0 μl, and dilute water to 10.0 μl in the test tube. Incubate for 4 h in a 37 ° C incubator.
4. Enzymatic cleavage product electrophoresis typing: 6% polyacrylamide gel electrophoresis (T=6%, C=3.3%, gel size: 82 mm×64 mm×0.75 mm). The electrode buffer was 1 x TBE. Electrophoresis products with 1/5 volume of loading buffer were electrophoresed, 220 v voltage, electrophoresis for 2-3 h.
5. Silver staining The gel was peeled off into a dyeing tray, fixed with 10% glacial acetic acid for 30 min, the fixing solution was removed, and the gel was rinsed 3 times with distilled water (within 2 min). Pour 200ml of 0.1% silver nitrate solution into the dyeing tray, stain for 30min, pour off the staining solution, and rinse the gel once quickly (within 20s). The color developing solution 200ml (3% Na2CO3, containing 0.05% formaldehyde, 2‰Na2S2O3) was poured into the dyeing tray and oscillated continuously until the band showed clear. Stop the color development with a fixative. Wash once with distilled water and stick to filter paper to dry and store.
[Result judgment]
The PCR product was 379 bp in length and the rs17750303 site was located at 140 bp, which was an A/C mutation. The rs837690 locus is located at 175 bp and is an AG mutation. Both sites are BstN I enzyme recognition sites, which can produce 1-4 (6 species) DNA fragments after digestion, and 10 profiles (genotype groups) can be found according to the number and length of the restriction fragments.
[Experimental discussion]
1. Note 1 The amplification product of the target fragment should be pure. If there are non-specific products (especially large fragments may contain enzyme recognition sequences), the enzyme activity will be competitive, and the sample will be incompletely digested or enzymatically digested; 2 enzyme The digestion process should be sufficient (ie, the ratio of substrate to enzyme should be appropriate, the digestion time should be guaranteed), and false negative results should be avoided; 3 the positive result of the enzyme digestion can determine the specific sequence detected, and the negative result can only indicate the non-enzyme recognition sequence, but not Accurately determine the specific sequence. 4 The enzyme recognition sequence, such as a methylated nucleotide, will not be cleaved.
2. Method evaluation The method is simple in operation and the result is easy to judge.
In this experiment, RFLP analysis was used to detect two SNP loci rs17750303 (A→C) and rs837690 (A→G) in the intron 3 of the GRIN2A subunit gene in the NMDA receptor.
[Experimental principle]
DNA restriction enzymes have an active function of recognizing a specific DNA sequence and cleavage of a DNA duplex at a specific site, and the DNA molecule is altered (or formed) by a mutation (nucleotide substitution, insertion or deletion). The enzyme recognition sequence prevents the DNA restriction endonuclease from (or can) cleave the target DNA fragment, and when the electrophoresis is detected, the original DNA fragment of the corresponding DNA restriction endonuclease recognition sequence becomes a short fragment; there is no corresponding DNA restriction The length of the original DNA fragment of the endonuclease recognition sequence will not change.
[experiment equipment]
PCR instrument, electrophoresis instrument, electrophoresis tank, microwave oven, incubator or constant temperature water bath.
[Experimental reagent]
Restriction enzyme BstNI, template DNA, Taq polymerase, PCR primer (upstream primer: 5-CTAAGCCTTCCCTTTATCACC-3'; downstream primer: 5-TTCACAGTCGTGCTTGTTTCT-3'), acrylamide, enzyme digestion buffer, electrode buffer, Load buffer, etc.
[experimental method]
1. PCR amplification: PCR reaction system is 20μl, containing template 2μl (about 50ng), primers 1.6μl, dNTP1.6μl (0.4mM), 10×Buffer buffer 2μl, Taq enzyme 0.5U, add double distilled water to 20.0 μl; PCR reaction conditions were 95 ° C 2 min, 94 ° C 30 sec / 64 ° C 30 sec / 72 ° C 30 sec (5 cycles), 94 ° C 30 sec / 63 ° C 30 sec / 72 ° C 30 sec (5 cycles), 94 ° C 30 sec / 60 °C30sec/72°C 25sec (23 cycles), 94°C 30sec/60°C 30sec/72°C for 1min.
2. Detection of PCR amplification products: 2 μl of the PCR amplification product was taken, and the amplification product of the target DNA fragment was detected by 1% agarose gel diving electrophoresis.
3. Enzyme digestion reaction: Take about 2.0 μl of the PCR product, add restriction endonuclease BstN I1U, 1.0×1× digestion reaction buffer 1.0 μl, and dilute water to 10.0 μl in the test tube. Incubate for 4 h in a 37 ° C incubator.
4. Enzymatic cleavage product electrophoresis typing: 6% polyacrylamide gel electrophoresis (T=6%, C=3.3%, gel size: 82 mm×64 mm×0.75 mm). The electrode buffer was 1 x TBE. Electrophoresis products with 1/5 volume of loading buffer were electrophoresed, 220 v voltage, electrophoresis for 2-3 h.
5. Silver staining The gel was peeled off into a dyeing tray, fixed with 10% glacial acetic acid for 30 min, the fixing solution was removed, and the gel was rinsed 3 times with distilled water (within 2 min). Pour 200ml of 0.1% silver nitrate solution into the dyeing tray, stain for 30min, pour off the staining solution, and rinse the gel once quickly (within 20s). The color developing solution 200ml (3% Na2CO3, containing 0.05% formaldehyde, 2‰Na2S2O3) was poured into the dyeing tray and oscillated continuously until the band showed clear. Stop the color development with a fixative. Wash once with distilled water and stick to filter paper to dry and store.
[Result judgment]
The PCR product was 379 bp in length and the rs17750303 site was located at 140 bp, which was an A/C mutation. The rs837690 locus is located at 175 bp and is an AG mutation. Both sites are BstN I enzyme recognition sites, which can produce 1-4 (6 species) DNA fragments after digestion, and 10 profiles (genotype groups) can be found according to the number and length of the restriction fragments.
The rs17750303-rs837690 locus genotype combination was determined according to the number and length of the fragment.
RFLP electrophoresis pattern of GRIN2A gene rs17750303 and rs837690 locus
1. DNA length standard; 2. PCR amplification product; 3. AA-AA type; 4. CC-AG type; 5. AC-AA type; 6. AC-GG type, 7. CC-AA type ; 8.CC-GG type; 9.AA-GG type; 10.CA-AG type.[Experimental discussion]
1. Note 1 The amplification product of the target fragment should be pure. If there are non-specific products (especially large fragments may contain enzyme recognition sequences), the enzyme activity will be competitive, and the sample will be incompletely digested or enzymatically digested; 2 enzyme The digestion process should be sufficient (ie, the ratio of substrate to enzyme should be appropriate, the digestion time should be guaranteed), and false negative results should be avoided; 3 the positive result of the enzyme digestion can determine the specific sequence detected, and the negative result can only indicate the non-enzyme recognition sequence, but not Accurately determine the specific sequence. 4 The enzyme recognition sequence, such as a methylated nucleotide, will not be cleaved.
2. Method evaluation The method is simple in operation and the result is easy to judge.
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