Section 3: Genetics, Cellular and Molecular Biology
Recombinant DNA technology
Subtopic: Fundamentals of Recombinant DNA technology
Question Statement:
The DNA sequence shown below is to be amplified by PCR:
5 ' GCTAAGATCTGAATTTTCC.....TTGGGCAATAATGTAGCGC3'
3' CGATTCTAGACTTAAAAG.....AACCCGTTATTACATCGCG5'
Options:
(A) Which one of the following pair of primers can be used for this amplification?
(B) 5' GGAAATTCAGATCTTAGT3' and 5' TGGGCAATAATGTAGCGC3'
(C) 5 ' GCTAAGATCTGAATTTTC3' and 5' GCGCTACATTATTGCCCA3'
(D) 5' CGGAAATTCAGATCTTAG3' and
5' GCGCTACATTATTGCCCA3'
5' GCTAAGATCTGAATTTTC3' and 5' TGGGCAATAATGTAGCGC3'
Official Answer Key:
B
Step-by-Step Solution & Explanation
### **Official Solution & Concept Analysis (GATE BT 2020 Q.37)**
**1. Governing Concept**:
- **Topic**: Molecular Biology & PCR Primer Design Rules
- To amplify double-stranded target DNA by Polymerase Chain Reaction (PCR):
- **Forward primer**: Anneals to the $3'$ end of the antisense (bottom) strand; its sequence is identical in the $5' \rightarrow 3'$ direction to the $5'$ end of the sense (top) strand.
- **Reverse primer**: Anneals to the $3'$ end of the sense (top) strand; its sequence is the **reverse complement** of the $3'$ end of the top strand.
- For top strand sequence:
$5'\text{-ATGCGAT...[middle]...GCTAGC-3'}$
The reverse primer must be $5'\text{-GCTAGC-3'}$ written in $5' \rightarrow 3'$ orientation as the reverse complement of the 3' terminus.
**2. Option Analysis**:
- **Option (A) is INCORRECT**: Uses direct top strand sequence instead of reverse complement.
- **Option (B) is CORRECT**: Formally matches the correct $5' \rightarrow 3'$ forward and reverse complement primer sequences.
- **Option (C) is INCORRECT**: Inverted primer orientations.
- **Option (D) is INCORRECT**: Non-hybridizing primer sequences.
**3. Final Answer**:
- **Correct Choice**: **B**
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