Question

Which of the following is the correct sequence for restriction fragment length polymorphisms (RFLP) steps sequence?
A. Probing hybridization of separated DNA.
B. Blotting of digested DNA onto a membrane.
C. Isolation and restriction digestion of the DNA.
D. Visualization by autoradiography.

• A. A, B, C, D
• B. C, B, A, D
• C. B, A, D, C
• D. C, B, D, A

Hint:

Remember the RFLP/Southern Blotting sequence with the acronym \textbf{DBAV}: \textbf{D}igest \(\rightarrow\) \textbf{B}lot \(\rightarrow\) \textbf{A}dd Probe (Hybridize) \(\rightarrow\) \textbf{V}isualize.

Answer 1

The Correct Option is B

RFLP is a multi-step DNA analysis technique. Let's arrange the steps in logical order.
Step 1: Prepare the DNA. The process must start with the DNA itself.
C. Isolation and restriction digestion of the DNA: First, DNA is extracted from the sample. Then, restriction enzymes are used to cut the DNA into fragments of different sizes. (After this, the fragments are separated by size using gel electrophoresis, which is an implicit step).

Step 2: Transfer the DNA. The DNA fragments from the gel must be transferred to a more stable medium for analysis.
B. Blotting of digested DNA onto a membrane: The separated DNA fragments are transferred from the gel to a nylon membrane in a process called Southern blotting.

Step 3: Label the DNA. Specific fragments of interest must be identified.
A. Probing hybridization of separated DNA: A radioactive or fluorescent probe (a short, known DNA sequence) is added. It binds (hybridizes) to its complementary sequence among the DNA fragments on the membrane.

Step 4: Detect the Labeled DNA. The location of the bound probe must be made visible.
D. Visualization by autoradiography: If a radioactive probe was used, the membrane is placed against an X-ray film. The radioactivity exposes the film, creating a band pattern that reveals the location of the specific DNA fragments.
The correct sequence is C \(\rightarrow\) B \(\rightarrow\) A \(\rightarrow\) D.