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Southern blotting application - Assignment Example

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Restriction Fragment Length Polymorphism (RFLP) is a technique that involves the detection of differences in homologous DNA sequences by the presence of different lengths of fragments after digestion of the DNA samples in question with specific restriction endonucleases. RFLP is…
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Southern blotting application
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Download file to see previous pages A labeled DNA sequence is usually an RFLP probe that hybridizes with either a single or multiple fragments of the digested DNA sample after separation by gel electrophoresis resulting to showing a unique blotting pattern attributed to a genotype that is specific to a locus. RFLP probes are usually short, single or low copy genomic DNA or cDNA clones. Application of RFLP probes are usually used in variation analysis such as in paternity tests, ping and diagnosis of hereditary diseases. RFLP probes are also used in genome mapping.
RFLPs are detected by using restriction enzyme that can recognize and fragment a sample of DNA where there is a specific short sequence. This step is known as restriction enzyme digestion. Single nucleotide polymorphisms can either create or destroy restriction endonuclease recognition sites therefore affecting the length and the quantity of DNA fragments resulting from restriction enzyme digestion.
Agarose gel electrophoresis separates the resulting DNA fragments and transferred to a membrane through a Southern blot procedure. The membrane is a paper like matrix made up of nitrocellulose and usually has a slight positive charge. The fragment lengths that are complementary to the RFLP probes and are considered as alleles in genetic analysis is determined by the hybridization of the membrane to the labeled DNA probe (National Center for Biotechnology Information ).
In developing RFLP probes, a methylation sensitive enzyme digests the total DNA thus enriching the library for sequences that are single or low copy expressed. The digested DNA is size fractionated on preparative agarose gel and excision, elusion and cloning into plasmid vector of is done. The digested plasmids are consequently screened for inserts. Total sheared DNA can be used to probe southern blots of the inserts to select clones that can hybridise to single and low copy sequences. Genomic DNA of different genotypes ...Download file to see next pagesRead More
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