Q.50 The correct sequence of the Southern blotting procedure, as applied to DNA fingerprinting is
A. Incubation of nylon membrane with radiolabelled DNA probe.
B. Separation of digested DNA fragments by agarose gel electrophoresis.
C. Denaturation of separated DNA and transfer to nylon membrane.
D. Exposure of X-ray film to membrane for analysis of results.
E. Digestion of chromosomal DNA with restriction endonucleases.
Choose the correct answer from the options given below :
(1) E, D, C, B, A
(2) E, B, C, A, D
(3) A, B, C, D, E
(4) A, C, B, E, D
The correct sequence of Southern blotting is E → B → C → A → D (option 2).
Question Breakdown
Southern blotting detects specific DNA sequences in DNA fingerprinting via restriction digestion, electrophoresis, transfer, hybridization, and detection. The logical workflow follows sample preparation to visualization.
Option Analysis
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(1) E, D, C, B, A: Wrong; X-ray exposure (D) can’t precede electrophoresis (B).
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(2) E, B, C, A, D: Correct → Digestion → Electrophoresis → Denature/transfer → Probe hybridization → Autoradiography.
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(3) A, B, C, D, E: Wrong; probe incubation (A) requires prior DNA separation/transfer.
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(4) A, C, B, E, D: Wrong; starts with hybridization before DNA preparation.
Detailed Steps (E-B-C-A-D)
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E: Restriction endonucleases cut chromosomal DNA at specific sites (RFLPs).
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B: Agarose gel separates fragments by size (smaller migrate faster).
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C: DNA denatured (NaOH) → ssDNA transferred to nylon via capillary action.
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A: Radiolabeled probe hybridizes to target sequences (VNTRs in fingerprinting).
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D: X-ray film detects radioactive bands → unique DNA profile.
Southern blotting procedure sequence DNA fingerprinting follows E→B→C→A→D (digestion→electrophoresis→transfer→probe→autoradiography), essential for GATE Life Sciences molecular diagnostics. Alec Jeffreys’ technique revolutionized forensics/paternity testing via VNTR patterns.
Step-by-Step Protocol
| Step |
Code |
Process |
Purpose |
| 1 |
E |
Restriction digestion |
Generate RFLP fragments |
| 2 |
B |
Agarose electrophoresis |
Size separation |
| 3 |
C |
Denature + Southern transfer |
Immobilize ssDNA |
| 4 |
A |
Radiolabeled probe hybridization |
Sequence-specific detection |
| 5 |
D |
X-ray film exposure |
Visualize DNA fingerprint |
Fingerprinting Application
VNTR probes create individual barcodes (13 CODIS loci in forensics). Probability of two people matching: 1 in 10¹⁸.
Exam Strategy
GATE tests workflow logic: Preparation→Separation→Transfer→Detection→Visualization. Trap: Confusing probe hybridization timing. Memorize: “Digest→Run→Blot→Probe→Expose”. PCR replaced Southern in modern forensics but remains exam staple.
