Southern Blotting

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

2. The Southern blot is used to detect the presence of a particular piece of DNA as a sample.

3. The DNA detected can be a single gene, or it can be a part of a longer piece of DNA such

as a viral genome.

4. The key to this method is hybridization.

Hybridization: It is the process of forming a double stranded DNA molecule between a single stranded DNA probe and a single stranded target DNA.

There are two important features of hybridization.

a. The reactions are specific- the probes will only bind to target with a complementary sequence.

b. The probe can find one molecule of target in a mixture of millions of related but non-complementary molecules.

Steps of hybridization

i. The mixture of DNA molecule is separated by agarose gel electrophoresis.

ii. The molecules are immobilized on a matrix (the nitrocellulose membrane).

iii. The probe is added to the matrix to bind to the DNA molecules.

iv. Any unbound probes are then removed by washing with buffer.

v. The place where the probe is connected corresponds to the location of the immobilized target molecules.

8. Steps of Southern Blotting:

I. DNA Purification

 Isolate the DNA in question from the rest of the cellular material in the nucleus.

 Incubate the specimen with detergent to promote cell lysis.

 Lysis frees cellular proteins and DNA.

 Proteins are enzymatically degraded by incubation with proteinase.

 Organic or inorganic extraction removes proteins.

 DNA is purified from solution by alcohol precipitation.

 Visible DNA fibres are removed and suspended in buffer.

II. DNA fragmentation

 Cut the DNA into different sized pieces.

 Use restriction endonucleases (RE)

 Nucleases hydrolyze the bonds that connect bases within the strand, resulting in cleavage of the strand.

 They cleave the double stranded nuclei acid only at the specific points.

 This allows for specific sequences to be identified more rapidly.

 Fragments are now easily separated by agarose gel electrophoresis.

III. Gel Electrophoresis

 Agarose or polyacrylamide gel

 Gel is soaked in a buffer which controls the size of the pores.

 Standards should also be run.

 Nuclei acids have a net negative charge and will move from the left to the right. The larger molecules are held up while the smaller ones move faster. This results in a separation by size.

 Gels can be stained with ethidium bromide.

IV. Blotting

 Transfer the DNA from the gel to a solid support.

 The blot is usually done on a sheet of nitrocellulose paper or nylon.

 DNA is partially depurinated with dilute HCl which promotes higher efficiency transfer by breaking down fragments into smaller pieces.

 DNA is then denatured with an alkaline solution such as NaOH.

 This causes the double stranded to become single stranded.

 DNA is then neutralized with NaCl to prevent re-hybridization before adding the probe.

 Transferred by either electrophoresis or capillary blotting.

 Capillary blotting fragments are eluted from the gel and deposited onto the membrane by buffer that is drawn through the gel by capillary action.

 The blot is made permanent by:

i. Drying at ~800C.

ii. Exposing to UV radiation.

V. Blocking

 Buffer binds to areas on the blot not occupied by patient DNA.

 Blocks the empty sides from being bound during hybridization.

VI. Preparing the probe

 Small pieces of DNA used to find another piece of DNA.

 Must be labelled to be visualized.

 Usually prepared by making a radioactive copy of a DNA fragment.

VII. Hybridization

 The labelled probe is added to the blocked membrane in buffer and incubated for several hours to allow the probe molecules to find their targets.

VIII. Washing

 Excess probe will have bound non-specifically to the membrane despite the blocking reagents.

 Blots are incubated with wash buffers containing NaCl and detergent to the wash away excess probe and reduce background.

IX. Detection

Radioactive probes enable auto-radiographic detection.

Summary of the procedure

1. Extract and purify DNA from cells.

2. DNA is restricted with enzymes

3. Sort by electrophoresis

4. Denature DNA

5. Transfer of nitrocellulose paper

6. Block with excess DNA

7. Wash off unbound probe

8. Autoradiograph

Applications

1. Identify mutations, deletions and gene rearrangements.

2. Used in prognosis of cancer and in prenatal diagnosis of genetic diseases.

3. Leukemias

4. Diagnosis of HIV-1 and infectious disease.

 Every person has repeated sequences of base pairs which are called Variable Number Tandem Repeats (VNTRs). To find a particular VNTR, we use a radioactive version of the one in question. This pattern is known as a DNA fingerprint.

 The specific technique to find out specific genes present in DNA fragment having unique character is called finger printing. Such techniques includes PCR, Real Time PCR,etc.

 All such technique comes under DNA finger printing.

Applications of DNA finger printing include

 Paternity and Maternity Testing.

 Criminal Identification and Forensics

 Personal Identification.