A PRESENTATION ON SOUTHERN BLOTTING TECHNIQUE BY: Dr. TAHIRA ANJUM
SOUTHERN HYBRIDIZATION It is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. n Southern blotting combines agarose gel electrophoresis for size separation of DNA with methods to transfer the size separated DNA to a filter membrane for probe hybridization. n
n n Southern blotting method is named after its inventor, the British biologist Edwin Southern who developed this procedure at Edinburgh University in the 1970 s. Southern blotting could be used to locate a particular gene within an entire genome
Steps involved in southern blottingn n n 1. Digest the DNA with an appropriate restriction enzyme. 2. Run the digest on an agarose gel. 3. Denature the DNA (usually while it is still on the gel).
n n n 4. Transfer the denatured DNA to the membrane. Traditionally, a nitrocellulose membrane is used. 5. Probe the membrane with labeled ss. DNA. 6. Visualize radioactively labeled target sequence.
Resultn Hybridization of the probe to a specific DNA fragment on the filter membrane indicates that this fragment contains DNA sequence that is complementary to the probe.
POLYMERASE CHAIN REACTIONn . PCR is the amplification of a small amount of DNA into a larger amount. It is quick, easy, and automated. Larger amounts of DNA mean more accurate and reliable results for your later techniques
n The techniques was developed by Nobel Laureate Biochemist Kary Mullis in 1984 and is based on the discovery of the biological activity at high temperatures of DNA Polymerases found in Thermophiles (bacteria that live in hot springs).
To perform PCR on a sample we will need four thingsn n 1. The target sample 2. A primer 3. Taq polymerase 4. Nucleotides
There are three major steps to PCR and they are repeated over n over again n 1. Target sample is heated 2. Temperature is reduced and the primer is added. 3. New pieces of ss. DNA are made.
Applications of PCRn n n Isolation of genomic DNA Amplification and quantitation of DNA PCR in diagnosis of diseases
n The Southern blotting technique is extremely sensitive. It can be used to map the restriction sites around a single copy gene sequence in any genome (even of man). It is used for DNA fingerprinting, preparation of RFLP maps, detection and identification of the transferred genes in transgenic individuals, etc.