SOUTHERN BLOTTING

A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-

separated DNA fragments to a filter membrane and subsequent fragment

detection by probe hybridization.

The technique was developed by Sir E.M. Southern in 1975.

To identify a single gene among thousands of fragments of DNA and to detect sequences of DNA in an organism’s genome

Used in gene discovery and gene mapping

To analyze the genetic patterns in an organism’s DNA

To identify gene mutations, deletions, duplications, and gene rearrangements involved in diseases

To determine the number of copies of a particular DNA sequence presented in the genome of an organism

APPLICATIONS

To identify related DNA sequences in the genome and to determine if there is a gene family (a group of similar genes)

To detect certain cancers and genetic diseases, such as: Monoclonal leukemia populations Sickle cell mutations

Used in DNA fingerprinting, genetic engineering, & forensic science for tests such as: Paternity testing Personal identification Sex determination Species exclusion

RFLP Genome mapping

PRINCIPLEThe DNA mixture is separated by agarose electrophoresis that separates the DNA fragments by size, using an electric charge.

On completion of electrophoresis, a nylon membrane, which binds DNA, is placed on the gel ,and by using capillary action the DNA migrates into the membrane.

For nylon membranes, the DNA is cross-linked to the membrane by the action of ultraviolet light; nitrocellulose membranes use heat to fix the DNA to the membrane.

•For the detection of specific DNA sequences, a hybridization probe is used.•A hybridization probe is a short (100-500bp), single stranded nucleic acid that will bind to a complementary piece of DNA, or RNA in the case of Northern blots. •Hybridization probes are labeled with a marker so that they can be detected after hybridization. •The labels used include radioactivity, usually radioactive phosphorous, or fluorescent labels.•The excess probe is washed away and the attached probes detected. The results show which DNA fragments contain the sequence of interest.

CHEMICAL SOLUTIONS REQUIRED

1. Saline Sodium Citrate(SSC) 20X: 3 M NaCl (175.5g/lit) , 0.3 M Sodium citrate dihydrate(88g/lit.), pH adjusted to 7 using citric acid, volume made up to 1 litre.

2. 0.2 N HCl-Depurination Solution: Conc.HCl(11.5 N)-21.5 ml; Sterile water-978.5 ml.

3. Denaturation Solution: 1.5 M NaCl (87.75g), 0.5 M NaOH(20g); Volume made up to 1 litre.

4. Neutralisation Solution: 3 M NaCl(175.5g), 0.5 M Tris base(60.5g); Adjust pH to 8 with HCl , volume made up to 1 litre and autoclaved.

5. Hybridisation Buffer: 2%SSC+ add NaCl (AR grade) to conc.0.5 M; add SDS 1% slowly while stirring. Continue mixing on a magnetic stirrer.

6. Primary Wash Buffer: 2 M urea(120g), 0.1% SDS(1g); 50mM Sodium Phosphate, 150mM Nacl(8.7g), 1mM MgCl2(1ml), 0.2% blocking reagent (2g); volume made up to 1 lit

7. Secondary Wash Buffer: 20X stock; 1M Tris Base(121g) , 2M NaCl(112g), make up pH 10 then volume make up to 1 litre.

PROCEDURE

1.Agarose gel electrophoresis(1%) is performed on plant genomic DNA treated with a restriction nuclease of choice2. After visualising on UV, the gel is transferred to a tray of 500ml of depurination solution and place on rocker arm shaker for 10 minutes3.Acid is decanted, gel is rinsed in unionised water4.To the gel tray 500 ml denaturation solution is added and shaked on a rocker arm for 10 minutes.5. Denaturation solution is decanted, 500ml neutralisation solution is added and shaken for 10 minutes.6. A glass plate is placed on tray and a wick(Whatman 3mm paper, 2 cm wider than the width of gel and 35 cm long). Then add 300ml 10X SSCinto the tray such that the ends of the wick is dipped in the solution. No air must be present between the wick and plate

7. Gel taken from neutralisation is placed inversely on wick. No air bubbles must exist between wick and gel8. Nylon membrane placed on top of gel9. About 5 pieces of Whatman 3 mm paper is placed on the membrane,then glass plate of 500g is placed on it as a weight.10. Left for transfer for 18 hours overnight.11. After blotting,the transfer setup is dismantled and the membrane rinsed in 2% SSC and left to dry.12. Permanent immobilisation of DNA on membrane(on nitrocellulose by baking at 80°C in vacuo , on nylon by UV irradiation)13. First,the membrane is prehybridized in a solution designed to block the unused DNA binding sites on the membrane surface(0.2% Ficoll or BSA or PVP) to stop non-specific binding of probe.

14. Hybridization is carried out in a high-salt buffer containing a detergent, usually 2% SSC+1% SDS.15.DNA is treated with a solution of radioactively labelled probes which bind to the complementary sequences.16. Hybridization step must be carried out at a ‘stringency’ that results in the specific probe–target hybrid remaining stable while all other hybrids are unstable,the stringency being determined by the composition of the hybridization buffer and the temperature at which the experiment is carried out.17. Specificity is then achieved by a series of washes at increasing temperatures so that, hopefully,only the desired hybrid remains at the end of the procedure.18. After washing,the membrane is subjected to the detection procedure appropriate for the label that has been used,for example autoradiography for a radioactive label.

CONCLUSION

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