Restriction fragment length polymorphism (RFLP) analysis
Restriction fragment length polymorphism (RFLP) analysis
Restriction fragment length polymorphism (RFLP) is one of the most important analytical methods in molecular biology and is mainly used to (1) detect DNA sequence polymorphism and (2) explore gene diversity.
Operation method
Restriction fragment length polymorphism (RFLP) analysis
Principle
1. DNA restriction endonuclease has the active function of recognizing specific DNA sequences and cutting DNA double strands at specific sites. When DNA molecules change (or form) restriction endonuclease recognition sequences due to mutations (nucleotide substitutions, insertions, or deletions), DNA restriction endonuclease is unable to cut off the target DNA fragments (or can cut off the target DNA fragments), and when electrophoresis is detected, the length of the original DNA fragments becomes short fragments in the case of the presence of the corresponding DNA restriction endonuclease recognition sequences. When electrophoresis is performed, the original DNA fragments will become short fragments if the corresponding DNA restriction endonuclease recognition sequence is present, and the length of the original DNA fragments will remain unchanged if the corresponding DNA restriction endonuclease recognition sequence is not present. 2. Different bands are formed by analyzing these fragments through gel electrophoresis, and then southern hybridization and radiography are performed with the cloned DNA probes to obtain the RFLP profiles reflecting the specificity of an individual. Genomic DNA digested by restriction endonucleases produces fragments that differ in length.
Materials and Instruments
Template DNA Move 1.PCR扩增：PCR反应体系为20μl，含模板2μl（约50ng），引物各1.6μl，dNTP1.6μl（0.4mM），10×Buffer缓冲液2μl，Taq酶0.5U，加双蒸水至20.0μl；PCR反应条件为95℃2min，94℃30sec/64℃ 30sec/72℃30sec (5 cycles), 94℃30sec/63℃30sec/72℃30sec (5 cycles), 94℃30sec/60℃30sec/72℃25sec (23 cycles), 94℃30sec/60℃30sec/72℃1min. 2. Detection of PCR amplification products: Take 2μl of PCR amplification products and detect the amplification products of target DNA fragments by diving electrophoresis on 1% agarose gel. 3. Enzyme digestion: Take 2.0 μl of PCR product, add restriction endonuclease BstN I1U, 1.0 μl of 10× enzyme digestion buffer, and make up to 10.0 μl with evaporated water in a test tube. incubate for 4 h at 37℃. 4. Electrophoretic typing of digested products: 6% polyacrylamide gel electrophoresis (T=6%, C=3.3%, gel size 82mm×64mm×0.75mm). Electrode buffer is 1×TBE. 1/5 volume of the digested product will be added with sampling buffer for electrophoresis, 220 v voltage, electrophoresis for 2--3h. 5. 5. Strip the gel into the staining plate, fix it with 10% glacial acetic acid for 30 min, remove the fixative, and rinse the gel with distilled water for 3 times (within 2 min). Pour 200ml of 0.1% silver nitrate solution into the staining plate, staining for 30min, pour off the staining solution, distilled water quickly rinse the gel 1 time (within 20s). 200 ml of color development solution (3% Na2CO3, containing 0.05% formaldehyde, 2‰ Na2S2O3 ) was poured into the staining plate and shaken continuously until the band was clear. The color development was terminated with fixing solution. Wash once with distilled water, stick on filter paper to dry and store. Caveat 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) will compete for the enzyme activity, so that the sample digestion is incomplete or the enzyme digestion of stray bands;2. The enzyme digestion process should be sufficient (i.e., the ratio of substrate to enzyme should be appropriate, and the digestion time should be guaranteed) to avoid false-negative results;3. Positive results of enzyme digestion can determine the specific sequences detected, while negative results can only indicate non-enzymatic recognition of sequences, but cannot accurately determine the specific sequences.4. Enzyme-recognized sequences with methylated nucleotides will not be cut. Common Problems 1. Because changes in base-to-base substitutions, rearrangements, deletions, etc. between alleles of different individuals can lead to changes in restriction endonuclease recognition and cleavage resulting in differences in restriction fragment length between genotypes. 2. Extraction of DNA from different individuals: (1) Lysis of cells using appropriate chemicals or grinding of cells from broken tissues with a tissue homogenizer; (2) Digestion of most of the proteins and RNA with proteases and RNA enzymes; (3) Removal of proteins by extraction with organic reagents such as phenol and chloroform. For more product details, please visit Aladdin Scientific website.
Restriction endonuclease BstNI Taq polymerase PCR primers Acrylamide Enzyme digestion buffer Electrode buffer Sampling buffer
PCR Amplification Instrument Electrophoresis Instrument Electrophoresis Bath Microwave Oven Constant Temperature Oven Constant Temperature Water Bath