32. Choose the appropriate pair of primers to amplify the following DNA fragment by the polymerase chain reaction (PCR).
5′–GACCTGTGG-----------------------------ATACGGGAT–3′ 3′–CTGGACACC-----------------------------TATGCCCTA–5′
Primers
P. 5′–GACCTGTGG–3′
Q. 5′–CCACAGGTC–3′
R. 5′–TAGGCCATA–3′
S. 5′–ATCCCGTAT–3′
(A) P and R
(B) P and S
(C) Q and R
(D) Q and S
PCR primers must be designed to anneal specifically to opposite strands at the 3′ ends of the target DNA fragment for amplification. The correct pair binds to the forward (top) strand’s 5′ end and the reverse (bottom) strand’s 3′ end (or its complement).
Primer Binding Rules
PCR uses two primers: one matching the 5′ end of the sense strand (forward primer) and one matching the 5′ end of the antisense strand (reverse primer, complementary to the 3′ end of the sense strand). For the given fragment:
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Top strand (5’→3′): GACCTGTGG…ATACGGGAT
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Bottom strand (3’→5′): CTGGACACC…TATGCCCTA (5’→3′ equivalent: ATACGGGAT…CCACAGGTC reversed)
Primer P (5′-GACCTGTGG-3′) matches the top strand’s 5′ end exactly. Primer S (5′-ATCCCGTAT-3′) matches the bottom strand’s 3′ end (complement of ATACGGGAT is ATCCCGTAT).
Option Analysis
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(A) P and R: P binds forward correctly, but R (5′-TAGGCCATA-3′) mismatches the bottom strand’s end (should be ATCCCGTAT, not TAGGCCATA). No amplification.
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(B) P and S: P binds top 5′ end; S binds bottom 3′ end perfectly. This amplifies the fragment.
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(C) Q and R: Q (5′-CCACAGGTC-3′) matches bottom 5′ end (reverse primer location), R mismatches. Wrong orientation.
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(D) Q and S: Both bind bottom strand ends; no forward primer for top strand.
Correct answer: (B) P and S.
Introduction to PCR Primers Amplify DNA Fragment
Polymerase Chain Reaction (PCR) relies on primers that flank the target DNA fragment for specific amplification. In this MCQ, select the right primer pair from P, Q, R, S to amplify 5′–GACCTGTGG—————————–ATACGGGAT–3′ and its complement. Understanding PCR primers amplify DNA fragment principles ensures success in biotech exams and lab work.
PCR Primer Design Basics
Effective PCR primers are 18-30 nt long, 40-60% GC, and bind antiparallel to template strands. Forward primer matches the 5′ end of the top strand; reverse primer is complementary to the 5′ end of the bottom strand (anneals to 3′ end of top strand).
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Avoid self-complementarity to prevent dimers.
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3′ end should clamp with G/C for stability.
Analyzing Primer Options
| Option | Primers | Binding Analysis | Amplifies? |
|---|---|---|---|
| A | P & R | P: Top 5′ match R: Mismatch (TAGGCCATA ≠ ATCCCGTAT) |
No |
| B | P & S | P: Top 5′ exact S: Bottom 3′ complement |
Yes |
| C | Q & R | Q: Bottom 5′ R: Mismatch |
No |
| D | Q & S | Both on bottom strand | No |
Option B works as P extends rightward, S leftward from opposite strands.
Application in Molecular Biology
Mastering PCR primers amplify DNA fragment skills aids cloning, diagnostics, and genomics. Tools like Primer3 optimize designs; always verify complementarity manually for exams.
