Question

Following are the steps of the southern blot procedure.
 (i) Autoradiography
 (ii) Hybridisation with radioactive nucleic acid (probe).
 (iii) Blotting
 (iv) DNA fragments are treated to make them single-stranded
 (v) Electrophoresis
 (vi) Clearing of DNA by a restriction endonuclease.
 (vii) Isolation of DNA from sample

The correct sequence is:
A) (i), (ii), (iii), (iv), (v), (vi), (vii)
B) (vii), (vi), (v), (iv), (iii), (ii), (i)
C) (i), (ii), (vi), (vii), (iii), (iv), (v)
D) (vii), (vi), (i), (ii), (v), (iii), (iv)

Answer 1

Hint: Southern blotting is a DNA hybridization technique to locate a specific DNA sequence of interest, in a sample or test DNA. It is named after Edwin M. Southern.

Complete step-by-step answer:
First of all the high molecular weight DNA is digested using restriction endonucleases. Then the DNA fragments are electrophoresed on an agarose gel to separate fragments of different lengths. If the DNA fragments are still larger than 15 kb, then it is treated with dilute HCl as HCl depurinated DNA and reduces its size. The double-stranded DNA is then denatured by keeping it in an alkaline medium.
Then DNA is transferred to a positively charged nylon or nitrocellulose membrane (by suction or by putting some weight on the membrane and the gel since the nucleotides are negatively charged, they bind to the membrane). The nitrocellulose membrane is baked at 80 degrees to immobilise the DNA. The immobilised DNA is exposed to the hybridization probe or sequence complementary to the DNA of interest, which has been labelled. The hybridization probe will bind to any complementary sequence present in the test DNA. The membrane is then washed with an appropriate buffer to remove any unbound probe. After that autoradiography is done that detects radioactivity. The site of fluorescence represents the complementary DNA sequences present in the test DNA.
Hence the correct answer is option B.



Note: The principle of southern blot involves Hybridisation of a labelled complementary oligonucleotide (to the sequence of interest) sequence to the denatured target DNA. The binding is detected using an autoradiogram, which helps to identify the presence of the DNA sequence of interest.