Q.62 The following is the list of steps involved in cloning of a gene.
Which option represents the correct sequence of the process ?
(A). Transformation with blue and white selection.
(B). RFLP analysis.
(C). Isolation of plasmid DNA.
(D). Ligation of gene, into TA-cloning vector.
(E). Amplification of gene by PCR, using Taq DNA polymerase.
Choose the correct answer from the options given below:
1. (E), (D), (C), (B), (A).
2. (E), (B), (D), (C), (A).
3. (C), (E), (D), (B), (A).
4. (C), (B), (D), (A), (E).
Gene Cloning Sequence: Correct TA Cloning Order
The correct sequence for TA cloning is option 1: (E), (D), (C), (A), (B) – PCR amplification first, then ligation into TA vector, plasmid isolation, transformation with screening, and RFLP verification.
TA Cloning Workflow
TA cloning exploits Taq polymerase’s non-templated A-overhangs on PCR products ligating into T-overhang vectors (e.g., pGEM-T), bypassing restriction digestion for speed.
Blue-white screening (lacZα) distinguishes recombinants (white) from self-ligated vector (blue).
RFLP confirms insert identity post-cloning via diagnostic digests.
Correct Sequence: Option 1
E → D → C → A → B follows logical TA cloning protocol.
| Step | Code | Purpose |
|---|---|---|
| 1 | (E) | PCR amplification generates A-tailed insert |
| 2 | (D) | TA ligation into T-vector |
| 3 | (C) | Isolate ligated plasmid DNA |
| 4 | (A) | Transformation + blue/white screening |
| 5 | (B) | RFLP analysis verifies correct clones |
Option Analysis
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Option 1 (E,D,C,A,B): Correct—PCR → ligation → plasmid prep → transform/screen → verify.
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Option 2 (E,B,D,C,A): Wrong—RFLP (B) can’t precede ligation/verification of unknown insert.
-
Option 3 (C,E,D,B,A): Wrong—Plasmid isolation (C) before PCR (E) illogical; source DNA needed first.
-
Option 4 (C,B,D,A,E): Wrong—PCR (E) last impossible; amplification precedes all cloning steps.
Why This Order Matters
1. PCR (E): Taq adds 3'-A overhangs naturally
2. Ligate (D): A-T base pairs directionally into vector
3. Isolate (C): Purify ligated plasmids
4. Transform (A): E.coli uptake + X-gal/IPTG screening
5. RFLP (B): BamHI/HindIII digest confirms insert size
RFLP post-cloning distinguishes true recombinants from PCR artifacts/false positives—essential validation step. Classic NEET biotechnology question testing protocol logic.
