(Image source: National Human Genome Research Institute) Extraction, purification, fragmentation, and separation of target DNAĭNA is extracted from the target source and is broken into small fragments using restriction endonuclease enzyme.įragmented DNA is then electrophoresed on an agarose gel to separate the fragments according to their molecular weights. DNA loading buffer, TBE Buffer for electrophoresis Steps involved in Southern blotting Southern blotting technique.Enzymes: restriction nucleases, RNase A.Cellulose nitrate or nitrocellulose membrane filter with uniform porosity.The probe also has a radioactive atom or a fluorescent dye label, that following hybridization, permits the DNA fragment of interest to be detected from different DNA fragments present on the membrane. The membrane is then treated with a small piece of DNA or RNA called a probe, which has a complementary sequence to the target DNA. Following separation, the double-stranded pieces of DNA are denatured into single strands within the gel and transferred from the gel onto a blotting membrane. The target DNA is broken into small fragments using restriction endonucleases and is separated by electrophoresis. Extraction, purification, fragmentation, and separation of target DNA.In addition, RFLP from homozygous organism (AA), heterozygous organism (AS), and homozygous organism (SS) is produced in various lengths by Mst II enzyme. So, Mst II is not able to recognize this mutated sequence (CCTGTGG). As we know, when adenine nucleotide in the sequence CCTGAGG mutates into thymine nucleotide, the sickle-cell disease appears. Mst II will not recognize and will not cut, if DNA has a mutation from sickle cell disease. Mst II cuts non-mutated (normal) beta globin DNA at a particular site, which is CCTNAGG (N is any base). The restriction enzyme called Mst II is used in the identification of sickle-cell disease. Generally, restriction enzymes, binding to DNA, slide along a DNA until they recognize specific sequences of nucleotides, where they start to perform their work, i.e. Restriction enzymes are the enzymes, which cut at specific sequence of nucleotides called restriction sites. Finally, RFLPs can be needed to identify genetic disease as well as disease carrier. Also, RFLPs are used to avoid inbreeding by the identification of heterogeneous parents. We use RFLPs in forensic labs to determine a criminal or we can use RFLPs to determine fatherhood. Restriction fragment length polymorphisms (RFLPs) are fragments of various sizes created by polymorphic region on DNA, when restriction enzymes cut (digest) DNA. Also, some radioactive elements can be absorbed by human’s body becoming incorporated in bones, and it is actually, very hard to eliminate an absorbed radioactive element from the body. For example, even a small amount can cause cancer (sometimes affecting DNA). We do not use radioactive probes to detect DNA in Southern Blot Analysis because radioactive isotopes (radioactive elements) can cause very dangerous effects on human’s organism when they are not handled properly. Generally, to perform the technique, separated DNA fragments obtained by electrophoresis are transferred to a filter membrane and then, detection by probe hybridization is made. The Southern blot analysis is a technique that allows determining specific DNA sequence in a sample of DNA. Southern Blot Analysis - Sickle Cell Diagnosis Lab
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