The DNA bound to membrane is then treated with labelled probe.Step VI: Hybridization with labelled probes The membrane is then treated with casein or Bovine serum albumin (BSA) which saturates all the binding site of membrane.After the DNA of interest bound on the membrane, it is baked on autoclave to fix in the membrane.The separated strands of DNA is then transferred to positively charged membrane nylon membrane (Nitrocellulose paper) by the process of blotting.The SDS gel after electrophoresis is then soaked in alkali (NaOH) or acid (HCl) to denature the double stranded DNA fragments.The desired DNA fragments is separated by gel electrophoresis.The number of fragments of DNA obtained by restriction digest is amplified by PCR.RE cuts the DNA at specific site generating fragments The DNA is fragmentized by using suitable restriction enzyme.Denaturation: Treating with HCl and NaOH.Gel electrophoresis: SDS gel electrophoresis.Restriction digest: by RE enzyme and amplification by PCR.The probes are labeled with a marker so that they can be detected after hybridization. A hybridization probe is a short (100-500bp), single stranded DNA.Separated DNA fragments after transferring on nylon membrane, the desired DNA is detected using specific DNA probe that is complementary to the desired DNA. Basically, the DNA fragments are separated on the basis of size and charge during electrophoresis.This technique is based on the principle of separation of DNA fragments by gel electrophoresis and identified by labelled probe hybridization. Southern blotting is a hybridization technique for identification of particular size of DNA from the mixture of other similar molecules. Southern blotting is an example of RFLP (restriction fragment length polymorphism).Southern blotting is a prerequisite to techniques such as restriction fragment length polymorphism (RFLP) analysis. Southern blotting is a technique which is used to confirm the identity of a cloned fragment or for recognition of a sub-fragment of interest from within the cloned DNA, or a genomic DNA. Southern hybridization helps to detect specific fragment against a background of many other restriction fragments. Separated by electrophoresis is transferred from gel to a membrane which in turn is used as a substrate for hybridization analysis employing labeled DNA or RNA probes specific to target fragments in the blotted DNA. Southern blotting involves separation of restricted DNA fragments by electrophoresis and then transferred to a nitrocellulose or a nylon membrane, followed by detection of the fragment using probe hybridization. It was the first blotting technique to be devised, named after its pioneer E.M Southern, a British biologist. Southern hybridization commonly known as southern blot is a technique employed for detection of a specific DNA sequence in DNA samples that are complementary to a given RNA or DNA sequence. The basic principle behind the southern hybridization is the nucleic acid hybridization. According to Watson-Crick base pairing, adenine binds with thymine and guanine binds with cytosine by hydrogen bonding. Nucleic acid hybridization is a basic technique in molecular biology which takes advantage of the ability of individual single-stranded nucleic acid molecules to form double-stranded molecules.
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