![]() Ĭredit: Oder Zeichner: abigail /Autoradiogram An interactive demonstration of the first DNA fingerprinting. The probe binds selectively to complementary sequences to reveal a series of distinct bands. Following transfer of the DNA onto a membrane through capillary action, the membrane is probed with radioactive probe DNA. Digestion of genomic DNA will result in a large smear. To visualize this probe hybridization, a film is exposed to the filter and processed.įollowing restriction digestion, the samples are resolved on an agarose gel. Following a lengthy transfer, the filter is denatured to and incubated with the radioactive probe. A filter is placed on top of the agarose and pressed against it to transfer the DNA in a process called Southern Blotting. Upon resolving on an agarose gel, genomic DNA that does not hybridize with the probe will obscure the locus of interest as a large smear. This probe will be able to bind to both fragments given sufficient sequence overlap. Note that the probe overlaps a restriction site in one of the alleles. The schematic below illustrates a comparison of restriction profiles between two sources. The presence or absence of a restriction site may arise from single nucleotide polymorphisms ( SNPs) that reveal the natural variation between people. In this case, the phenotype for these alleles is the actual sequence that may or may not contain restriction sites. However, these loci may contain different alleles. Since people are 2N, they have pairs of homologous chromosomes with the same loci. RFLP analysis was designed for forensic science to discriminate between people. the rectangle sitting on the chromosomes correspond to a probe locus. Homologous chromosomes with restriction sites noted by triangles. Agarose gels would be transferred to a membrane or filter where they would be hybridized to these radioactive probes. RFLP analysis requires that a probe to a specific area of DNA be used to identify specific locations. In fact, the resulting agarose gel would simply show a large smear of DNA. However, if one were to take the entirety of the human genome and chop it up with a restriction enzyme, many indecipherable fragments would be made. Using Restriction Enzymes, these differences in sequences may be teased out. DNA from differing sources will have variations or polymorphisms throughout the sequence. Restriction fragment length polymorphism ( RFLP) is a technique that exploits variations in DNA sequences.
0 Comments
Leave a Reply. |