southern blotting (used to detect the presence of a particular piece of DNA in a sample)
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The technique was developed by E.M. Southern in 1975. The Southern blot is used to detect the presence of a particular piece of DNA in a sample mujahid hussain, Department of Botany, University of Sargodha, Sargodha, Punjab, Pakistan
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southern blotting (used to detect the presence of a particular piece of DNA in a sample)
- 1. Southern Blotting Department of Botany University of Sargodha, Sargodha Punjab, Pakistan mujahid.hussain7877@gmail.com 1
- 2. Southern blotting • The technique was developed by E.M. Southern in 1975. • The Southern blot is used to detect the presence of a particular piece of DNA in a sample. • The DNA detected can be a single gene, or it can be part of a larger piece of DNA such as a viral genome. mujahid.hussain7877@g mail.com 2
- 3. Steps • Extract and purify DNA from the cell • DNA is restricted with enzyme • Separated by gel electrophoresis • Denatured the DNA • transfer to the Nitrocellulose membrane paper (Blotting) • Add radio labelled probe for hybridization to take place • Wash off unbound probe • Autoradiograph mujahid.hussain7877@g mail.com 3
- 4. Isolation of DNA • Isolate the DNA from the rest of the cellular material in the nucleus. • Incubate specimen with detergent to promote cell lysis. • A buffer containing a detergent such as sodium dodecyl sulfate (SDS) is usually sufficient to disrupt the cell membranes and release high- molecular weight DNA. mujahid.hussain7877@g mail.com 4
- 5. Isolation of DNA • Lysis frees cellular proteins and DNA. • Proteins are enzymatically degraded by incubation with proteinase. • Digestion of RNA with ribonuclease. • A final treatment with ethanol, which precipitates the remaining nucleic acid polymers, enabling ribonucleotides and other low-molecular weight contaminants to be removed. mujahid.hussain7877@g mail.com 5
- 6. Restriction Digestion • Cut the DNA into different sized pieces. • Use restriction endonucleases (RE) • Example, Eco R1 • For genomic DNA, overnight digestion is usually required. mujahid.hussain7877@g mail.com 6
- 7. Gel electrophoresis • Nucleic acid have a net negatively charge and will move from left to right. • The larger molecule are hold up while the smaller ones moves faster. • This results in separation by size. • Agarose gels have microscopic pores • Gel is soaked in a buffer which controls the size of pores. • Gel can be stained with ethidium bromide. mujahid.hussain7877@g mail.com 7
- 8. Gel electrophoresis • EtBr causes the DNA to fluoresce under UV light. • This will help to know the exact migration of DNA. Denaturation and blotting . • DNA is then denature with an alkaline solution e.g. NaOH. • This causes the double stranded of DNA to single stranded. • The process of transferring the DNA from the gel to membrane is called as blotting. • The blot is done on the sheet of nitrocellulose membrane or nylon. • DNA is neutralize with NaCl to prevent re-hybridization before adding the probe. • Transfer is by capillary blotting. • The blot is made permanent either by • 1) Drying at 80~ C • 2)Exposing to UV irradiation. mujahid.hussain7877@g mail.com 8
- 10. Hybridization • It is a process of forming a double stranded DNA molecule between a single stranded DNA probe and a single stranded target DNA. • The labelled probe is add to the membrane in a buffer and incubated for several hours to allow the probe molecule to find their targets. • Probe • Small piece of labelled DNA used to find another piece of DNA. mujahid.hussain7877@g mail.com 10
- 11. Wash and autoradiography • Wash excess probe that are bound non-specifically with the membrane. • Blot is incubate with wash buffer containing NaCl and detergent to wash away excess probe. • Radioactive probes enable autoradiography detection. • The probe is radioactive, the particle it emits will expose X-ray film. • After development, there will the dark spots on the film where the probe bound. mujahid.hussain7877@g mail.com 11
- 13. Advantages • Examine entire genome • Easier • Detect multiple product • Disadvantages • Costly • Greater quantity of DNA required • Labor intensive • Time consuming • This technique requires fresh or frozen specimens mujahid.hussain7877@g mail.com 13
- 14. Application • To identify specific DNA in the sample • To isolate desired DNA for making of rDNA • Used to prognosis cancer and in prenatal diagnosis of genetic disease • Used in phylogenetic analysis • Diagnosis of HIV-I and infectious disease • And application DNA finger printing mujahid.hussain7877@g mail.com 14