Sunday, March 31, 2013

Southern Blotting / Hybridisation!

Southern blotting is used for finding specific DNA sequence from a DNA sample by using the combination of techniques like  gel electrophoresis, capillary electrophoresis and hybridization. The DNA from the gel (after gel electrophoresis)is transferred to a nitrocellulose membrane and hybridization analysis is done for finding out the sequence of our interest using a probe)

Let us discuss here the steps involved in doing Southern blotting. The steps are
1) Preparation of Sample
2) Restriction of the Sample
3) Gel Electrophoresis
4) Pre-treatment of the gel
5) Blotting
6) Hybridization

1) Preparation of Sample: (plant/animal/bacterial)

For tissue or blood samples the DNA could be obtained by treating with SDS (Sodium dodecyl Sulphate)
If we are going to have the DNA from a bacterial sample, there are separate protocols for extracting genomic DNA and pDNA. For extracting DNA from plants, CTAB (cetyltrimethyl ammonium bromide)
is used. This CTAB binds with the DNA and helps in better extraction using phenol. This CTAB is used specifically for plants, because they contain more carbohydrates than animal and bacterial cells.

2) Restriction of the Sample:

Specific restriction enzymes could be used fro restricting the sample of DNA.  Only one restriction enzyme can be used or more than one enzyme could also be used.  For restricting the sample of DNA containing 1 micro gram per microlitrethe following amount of buffer, enzyme could be used. (Choose the buffer corresponding to the enzyme you use) This volume could also be scaled up.

Components
Volume (micro litres)
10X buffer
1.0
DNA
1
Water
7.5
Enzyme
0.5
Total
10









3) Gel Electrophoresis: 
Agarose gel electrophoresis is the next step. The restricted sample is run in the agarose gel (% of the gel depends on the sample)

4) Pre-treatment of the gel:
The gel is pre-treated with 0.25 mol/litre HCl for 30 min and also treated with an alkali.
The reasons for pre-treatment:

  • Acid treatment helps in breaking the DNA in the bands formed in the gel into smaller fragments and this also leads to little depurination!
  • Alkali treatment is done for denaturing the DNA by breaking the "H" bonds. This helps the DNA in binding easily to the membrane during the blotting. And also this helps in the hybridization with the probe.
5) Capillary Blotting:

This could be done by using nitrocellulose membrane or nylon membrane.

i) Using nitrocellulose membrane (high salt transfer)

While using nitrocellulose membrane, gel must be neutralized after the alkali treatment because at the pH above 9, there won’t be efficient binding of DNA with the nitrocellulose membrane. The neutralization is done by soaking the gel in Tris – salt buffer.

Blotting is generally done using nitrocellulose membrane. The capillary electrophoresis is set up as shown in the figure using SSC buffer (20X) (SSC contains 3mol/litre Sodium chloride and 0.3 mol/litre Sodium citrate) blotting for about 18 hours.

After the blotting or transfer is over, the membrane is washed with SSC (2X) and then dried. In this case, the DNA won’t be firmly attached to the membrane, so, the membrane could be baked at 80degree C for 2 hours which will result in semi permanent attachment of the DNA to the membrane

This same procedure followed for the uncharged nylon membrane also, till the washing and drying. After that for strong binding of DNA with the membrane, the nylon membrane is UV treated.

ii) Using a positive charged nylon membrane:
In this method, a different buffer is used and no alkali pre treatment of the gel is needed. Here, O.4 mol/litre NaOH is used as buffer and this gives immediate covalent binding of the DNA to the membrane and hence no baking or UV treatment is needed.

Advantages of nylon membrane :
i)                    Better binding of DNA, even 50bp DNA binds efficiently whereas nitrocellulose membrane efficiently binds 500bp only.
ii)                   Nylon membranes cannot be damaged easily and hybridization could be done repeatedly upto 8 or 10 times.
iii)                 No need for post blotting baking or UV treatment for +ve charged nylon membranes and has reduced loss of DNA.

Electrophoresis or electroblotting from the gel to membrane could be done instead of capillary blotting and also vacuum blotting which draws the buffer through the gel to the membrane could also be done. This reduces the time of blotting to a few hours to minutes.

6)Hybridization:
Pre-hybridization step: This is done to block the areas of the membrane which donot contain DNA, to avoid random hybridization during hybridization. Generally this is done by treating the membrane in the buffer containing Salmon sperm DNA (popular choice of DNA used for blocking) for 15 minutes to 3hours depending on the membrane type.

Then actual hybridization is done by using high salt buffer with a detergent (2X SSC + 1% SDS) containing the probe DNA sequence. For increased sensitivity 10%Dextran sulphate or 8% polyethylene glycol 6000 is added to the buffer and probe mixture, this increases the cross linking between the probe and sample DNA. For increasing the specificity, the temperature at which the bonding between the probe and the sample DNA will be stable must be used; at this temperature all the other random bonding will be unstable.

Thus, we could identify the DNA fragment containing our interested sequence using Southern blotting.


Any queries? feel free to comment. 


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