What Is Restriction Fragment Length Polymorphism?
Restriction fragment length polymorphism (RF LP) is referred to as PCR-RFLP analysis. It is mainly to design appropriate amplification primers so that the amplified fragments include one or several polymorphic restriction endonuclease recognition sequences. After PCR amplification, the restriction enzyme is used to cut the PCR products. Enzymatic digestion (Amp-FLP) is highly polymorphic for repeats such as VNTR.STR due to the number of repeat units. Therefore, using specific primers on both sides of the repeat sequence for PCR amplification, the resulting amplified fragments are highly polymorphic. These alleles of different lengths can be separated by PAGE. [1]
Restriction fragment length polymorphism
- Restriction fragment length polymorphism (RF LP) is referred to as PCR-RFLP analysis. It is mainly to design appropriate amplification primers so that the amplified fragments include one or several polymorphic restriction endonuclease recognition sequences. After PCR amplification, the restriction enzyme is used to cut the PCR products. Enzymatic digestion (Amp-FLP) is highly polymorphic for repeats such as VNTR.STR due to the number of repeat units. Therefore, using specific primers on both sides of the repeat sequence for PCR amplification, the resulting amplified fragments are highly polymorphic. These alleles of different lengths can be separated by PAGE. [1]
- Restriction fragment length polymorphism
- The technology uses a restriction enzyme to recognize
- One is due to a single occurrence at the restriction enzyme site.
- different
- However, this linkage is incomplete even among people of the same race or in the same region. This is mainly manifested in two aspects: First, a certain RFLP haplotype is linked to a certain mutant gene, but the same haplotype can also be found in normal people. Therefore, using haplotypes cannot say with certainty whether an individual has the mutant gene. However, this linkage is different even among people of the same race or in the same region. Therefore, using haplotypes cannot say with certainty whether an individual has the mutant gene. Second, the same mutation can also be associated with several haplotypes. For these two cases, in order to make a prenatal diagnosis, you can first investigate the family by investigating a certain haplotype in the family, and then test the fetal haplotype to determine whether the fetus is It also contains this mutant gene. However, sometimes it is impossible to determine whether a certain haplotype is linked to a certain mutation through pedigree investigation. At this time, it is impossible to make a correct judgment on its fetus. This is also the disadvantage of RFLP for diagnostics.
- DNA and DNA fingerprints in hypervariable regions
- Human satellite DNA or satellite DNA is made up of several short DNA fragments (about 10bp) that are repeated many times. The composition and copy number of the repeats are different in different individuals and different positions of the genome. After extracting the genomic DNA of different individuals, use restriction enzymes whose cut points can recognize a sequence of 4 bases without cutting the repeat fragment to cut the genomic DNA on both sides of the repeat fragment, and then subject the sample to gel electrophoresis . Repeating fragments of different lengths (mainly due to different copy numbers of the repeating units) will be separated, and then hybridized with specific probes containing these repeating sequences to form individual-specific autoradiography, also known as DNA fingerprint.
- Atlas of DNA fingerprints
- Depends on the core sequence of the probe used (ie the repeat unit in the repeat). There are currently two types of probes used, namely probe 33.15. Its core sequences are AGAGGTGGGCAGGTGG, and 33.6, namely AGGGCTGGAGG. That is to say, these two sequences are repeated different times at different positions in the human genome, and in different individuals' genomes, the two core sequences are also repeated differently at corresponding positions. In this way, using one of these two probes to hybridize with a suitable enzyme-cut human genomic DNA fragment will result in different DNA fingerprints in different individuals, and the DNA fingerprints of probe 33.5 are also different. DNA fingerprints have cell stability and germline stability, are inherited according to Mendelian law, and have high heterozygosity. Heterozygosity can be understood as follows. For RFLP caused by point mutations, there are only two polymorphisms for a certain polymorphic tangent point, that is, the tangent point is (+) or the tangent point is not (-). For RFLPs caused by different lengths of repeats in the hypervariable region, the number of repeats of the core sequence at a certain position in the genome varies from individual to individual, such as 10 copies in individual A and 15 copies in individual B , And individual C may be 18 copies and so on. Therefore, the number of repeats of the core sequence at the same position in different individuals is polymorphic, not bimorphic. Even if the number of core sequences is the same at one position on the genome, and thus the length of the core sequence is the same, the number of repeats of the core sequence may be different at other positions in the genome.
- Restriction fragment length polymorphism