Question

An analysis of chromosomal DNA using the southern hybridization technique does not use
A.Electrophoresis
B.Blotting
C.Autoradiography
D.PCR

Answer 1

Hint: Hybridization is the process of formation of a dsDNA molecule between a ssDNA probe and a ssDNA target. Southern hybridization technique is utilized in molecular biology, immunogenetics and various other molecular methods for detecting or identifying the DNA of interest from a mixture of DNA samples or a specific base sequence within the strand of a DNA.

Complete answer:
The technique of Southern hybridization was developed by E.M. Southern in the year 1975. He was a molecular biologist and helped in analysing the related genes in a DNA restriction fragment. This technique involves transfer of electrophoresis-separate DNA fragments to nitrocellulose carrier membrane and then the detection of the target DNA fragment is done by probe hybridization. The steps involved are:
a.Extraction and purification of DNA from cells
b.Restriction Digestion or DNA Fragmentation
c.Separation by Electrophoresis
d.Depurination
e.Denaturation
d.Blotting
e.Baking
f.Hybridization
g.Washing of unbound probes
h.Autoradiograph


1.DNA is digested with restriction enzymes and separated by gel electrophoresis. A membrane of nylon is laid under the gel and the DNA is transferred to it, drawing liquid out of the gel, and the process is called blotting.
2.The blotted DNA is covalently attached to the nylon membrane by a brief exposure of the blot to UV light.
3.The membrane is bathed in a solution to denature the DNA from dsDNA to ssDNA.
4.Then a hybridization solution containing small amounts of ssDNA probe is labeled using fluorescent or radioactive molecules. The probe DNA forms a stable duplex with the DNA molecules on the membrane that are complementary to it only if the hybridization is performed correctly.
5.The unhybridized probe is then washed off and the remaining radioactive signal appears in a distinct band upon detection.
6.These bands represent the presence of a particular DNA sequence within the mixture of DNA fragments.

Hence the correct answer is OPTION(D)

Note: Southern blots are useful for detecting fragments larger than those amplified by PCR. This technique was invented before the PCR but got replaced by PCR as it is simple, provides good speed, and convenience. Other types of blotting techniques were invented after the development of Southern blot.