Question

What is the first step in the Southern Blot Technique?
A) Denaturation of DNA on the gel for hybridization with specific probe
B) Production of a group of genetically identical cells
C) Digestion of DNA by restriction enzyme
D) Isolation of DNA from a nucleated cell such as the one from the scene of crime

Answer 1

For the detection of specific DNA, RNA, and protein molecules there are many techniques that have been used in the biotechnological process. These molecules are detected for many social issues like to solve any crime case, to detect any diseases, or to identify the biological parents of a missing child and the southern blotting is one of such techniques.

Complete Answer:
- The southern blotting is a process that involves the transfer of DNA fragments separated from electrophoresis to the carrier membrane that is mostly nitrocellulose and the subsequent analysis of the target DNA sequence through probe hybridization.
- The hybridization is a process in which the between the single stranded target DNA the double stranded DNA is formed as the target DNA and the probe is complementary to each other.
- Now, let’s understand the steps involved in this technique; the extraction of DNA from the target cell through genomic DNA extraction and then purified, this is the very first step that involves the southern blotting technique.
- The next steps involve the digestion of DNA through restriction enzymes called restriction endonuclease into small fragments of DNA. Then the DNA is separated with the help of agarose gel electrophoresis in which the DNA fragments move towards the positive anode as DNA is negatively charged.
- Now, with help of HCL partial depurination is carried out, after that the DNA is denatured with an alkali like alkaline solution, this results in the denaturation of the DNA that make it suitable for hybridization.
- The next step is the blotting process in which the denatured fragment of DNA are transferred to the nitrocellulose membrane by placing the gel on the buffer saturated filter paper and then the nitrocellulose membrane over the gel and lastly some dry filter papers are place over the membrane.
- With the help of the capillary action the fragments pulled towards the nitrocellulose membrane result in the formation of gel print in the membrane.
- After this process the nitrocellulose membrane is removed and baked at 80 degree Celsius for 2-3 hours, in the next step, hybridization is carried out in which the DNA fragments are hybridized with the probe, the probe DNA is labeled with the help of radioactive molecules so that it get easy to detect, after hybridization the membrane is washed in buffer so that the unbound probe get removed and at last it is observed with the help of autoradiograph.

Thus, the correct option is ‘D’. Isolation of DNA from a nucleated cell such as the one from the scene of crime.

Note: The southern blotting is used for the detection of target DNA but remember there are two more blotting techniques; western blotting and northern blotting technique. The western blotting is used for the detection of specific protein molecules and the northern blotting is used for the detection of the RNA molecules.