SOUTHERN BLOTTING | an Overview, Principle, Steps and Application

SOUTHERN BLOTTING

 by Meet Patel

Introduction

In the mid-70s, Edward m. southern developed a technique for the detection of DNA fragments among their size separated by gel electrophoresis. This technique became famous as “southern blotting”. In this technique, gDNA is isolated and allowed with molecular scissors (restriction-endonuclease) which is an enzyme that cut DNA strand and produce DNA fragment of various size.  Fragments are then separated by gel electrophoresis based on their size. Then these fragments are transferred to the nitrocellulose membrane with the help of blotting. After the blotting step, allow it to hybridize with a radio-labeled probe (chemically synthesized oligonucleotides, having length 100-500 bp.) which are complementary with the desired DNA fragment. Then examine the membrane in autoradiograph.

Principle

The DNA fragment separated by gel electrophoresis, hybridize with a labeled probe on a nitrocellulose membrane. The probe is labeled with a marker (Marker like radio element or fluorescence.). So they can be detected after hybridization.

Methods

1.         Restriction digestion:

•   Take a 2 ml vial. Add buffer solution, DNA sample, restriction endonuclease, and nuclease-free water. ( restriction digestion system makes up as per the following table a.)
•  Allow it to react at 37 degrees Celsius for 1-2 hours in the incubator.

Component	Final concentration/amount
Nuclease free water	To 50 µL
Buffer	1 X
BSA	0.05 units/ µL
DNA template	1 µg
Restriction endonuclease enzyme	5-10 U per µg of DNA template(should not exceed 10% of reaction volume)

2.       Gel electrophoresis:

(image 1: sample loaded in well by micropipette. The DNA fragment separates based on their size.)

• The gel was cast between the glass plate. The glass plate is separated by a spacer and along one edge placed comb (top side).
•  Assemble the electrophoretic assembly.
•  Agarose solution was prepared by dissolving the appropriate weight in boiling electrophoresis buffer. Allow the solution to cool down at 60 to 65 degrees Celsius then pour in assembly and allow to sit for at least one hour.
•  After that remove comb fills the electrophoresis buffer.
• Sample prepared with 5% glycerol and dye. Mix it properly and add the sample solution to the wells of the gel.
• Connect the assembly with the battery and turn on the switch to allow separation to take place.
• When the tracker (dye) is far from 1cm of the bottom edge, turn off the switch and take out the gel from the assembly.

3.       Denaturation :

(image 2: Double-stranded DNA treated with an alkaline solution, because of high pH, DNA strand denatured and become single-stranded DNA.)

• After gel electrophoresis, put the gel in the alkaline ( NaOH ) or acid ( HCl ) solution which is dentures the dsDNA to produce ssDNA.

4.        Blotting  :

• Take an ass piece and fill it with blotting buffer. Kindly put gel on it. The nitrocellulose membrane is put on the gel to avoid the air bubble placed between gel and membrane.



(image 3: southern blotting assembly. 2x SSC buffer which helps DNA fragment to transfer to gel from nitrocellulose membrane.)



• Take 4-5 pieces of filter paper and put on nitrocellulose membrane and lastly put appropriate weight on top.

• The blotting buffer transfer the DNA fragment to gel from the nitrocellulose membrane.

5.        Fixation :

• After blotting, nitrocellulose membrane bake in an autoclave for 30 min or treated with casein/BSA solution to block DNA fragment on nitrocellulose membrane gene.

6.        Hybridization :

(image 4: Radiolabeled RNA probes are complementary with desired DNA fragment in appropriate condition.)

• Take hybridization buffer in a glass dish. Add labeled probe inappropriate concentration and volume.
• The probe is complementary with the tdesdesireddre DNA fragment and labeled with either radioactive element or fluorescence dye.
• Allow it for several hours. Then wash the membrane with washing buffer to remove the non-hybridized probe.

7.        Visualization :

(Image 5: DNA fragment separated on the gel with an invisible bend. After the blotting, nitrocellulose membrane sock in radiolabeled RNA probe which gives visible bend in autoradiograph.)

• Desire DNA fragments can be detected on the membrane by autoradiography in case of probe labeled with a radioactive element or fluorescence spectroscopy in case of probe labeled with fluorescence dye.

Applications

1.  It uses to detect DNA fragments in a given sample.
2.  It can use in DNA fingerprinting.
3. It can use in markers like RFLP.
4. It can use to identify the mutation.
5. It uses for the diagnosis of the disease-causing gene.

Reference

1.  Southern, Edwin Mellor. "Detection of specific sequences among DNA fragments separated by gel electrophoresis." Journal of molecular biology98.3 (1975): 503-517. PubMed PMID: 1195397.