Transcript Slide 1
Southern blot Background • a method to detect the presence of a DNA sequence in a DNA sample. • The method is named after its inventor, the British biologist Edwin Southern. • Combines agarose gel eletrophoresis for size separation of DNA with methods to transfer the size-separated DNA to a filter membrane for probe hybridization. • The key to this method is hybridization. • Hybridization-process of forming a doublestranded DNA molecule between a singlestranded DNA probe and a single-stranded target DNA. • There are two important features of hybridization: – The reactions are specific-the probes will only bind to targets with a complementary sequence. – The probe can find one molecule of target in a mixture of millions of related but noncomplementary molecules. DNA extraction Procedure DNA digestion Agarose electrophoresis Gel and membrane preparation Transfer Prehybridization Probe preparation and hybridization Wash Detection http://www.accessexcellence.org/AB/GG/southblotg.html DNA extraction • Quality and amount of DNA is important for southern blot. • Agarose electrophoresis check DNA integrity after extraction. High quality intact DNA should give the appearance of a single band. Degraded material will smear downwards. • Avoid RNA, protein, endogenous nuclease contamination. • Avoid DNA degradation. Enzyme digestion Restriction endonuclease digestion of genomic DNA is different from plasmid. 1ug genomic DNA: 5-30U enzyme Determine appropriate enzyme amount and digestion time. Avoid incomplete digestion: use large volume or use high concentrated enzyme. Transfer • Depurination is not required for DNA fragments <10kb in size. Do not over depurinate, 10mins(or until the bromophenol blue turns yellow) is usually sufficient for most samples. • DNA is then denatured with an alkaline solution such as NAOH,which causes the double stranded to become single-stranded. • DNA is then neutralized with NaCl to prevent rehybridization before adding the probe. • Transferred by either electrophoresis or capillary blotting. • Avoid trapping any air bubbles during transfer. • Large DNA fragments need overnight to 24h transfer. Pre-hybridization • 4-5 hours • Buffer binds to areas on the blot not occupied by patient DNA. • Blocks the empty sites from being bound during hybridization. Hybridization and Washing Lower hybridization temperatures achieve lower stringency. 65-68℃ is suitable for most long probes(>100base). Stringency washes will dependent on the nature ofn probe and target to be hybridized. The lower the salt concentration, the greater the stringency. The higher the washing temperature, the greater the stringency. Watch points • Using too little DNA-compromise the sensitivity of the test • Using too much DNA- poor restriction enzyme digestion • Using too high voltage setting for electrophoresisgel to melt or appearance of artifacts • Improper blocking-high background and uninterpretable results. • Insufficient washing-high background and uninterpretable results. • Excess washing- dissociate the specific hybrids.