Southern Blotting Sequence Explained

Southern Blotting is a molecular biology technique used to detect specific DNA sequences within a DNA sample. The correct sequence of steps is crucial for accurate results.

Steps in Southern Blotting

1. Fragmentation with Restriction enzyme (C): The genomic DNA is first digested using restriction enzymes. These enzymes cut the DNA at specific recognition sites, producing DNA fragments of various sizes.
2. Gel Electrophoresis (A): The DNA fragments generated in the previous step are then separated based on their size using gel electrophoresis. Smaller fragments migrate faster through the gel matrix than larger ones.
3. Transfer to nitrocellulose filter (D): After separation, the DNA fragments are transferred from the fragile gel onto a more durable membrane, such as a nitrocellulose or nylon membrane. This process is commonly referred to as 'blotting'.
4. Hybridization with radioactivity labelled probe (B): The membrane containing the immobilized DNA fragments is incubated with a labeled DNA probe. This probe is a single-stranded DNA sequence that is complementary to the target DNA sequence of interest. The probe binds (hybridizes) specifically to the target DNA on the membrane.
5. Autoradiography (E): If a radioactive probe is used, autoradiography is performed. The membrane is exposed to X-ray film, which detects the radiation emitted by the probe bound to the target DNA, revealing the location and size of the target DNA sequence.

Correct Sequence Confirmation

Based on the standard protocol, the correct sequence is:

• C. Fragmentation with Restriction enzyme
• A. Gel Electrophoresis
• D. Transfer to nitrocellulose filter
• B. Hybridization with radioactivity labelled probe
• E. Autoradiography

Therefore, the correct option is C, A, D, B, E.