Selecting Primers for DNA Amplification

Choose the pair of primers that will allow amplification of the DNA sequence both two given structures the DNA ?
5’GACCTGTGGAAGC…………..CATACGGGATTGA3′
3’CTGGACACCTTCG……………GTATGCCCTAACT5′
1. 5’GACCTGTGGAAGC3′
2. 5’CTGGACACCTTCG3′
3. 5’CATACGGGATTGA3′
4. 5’TCAATCCCGTATG3′

(a) 1 and 2
(b) 1 and 3
(c) 1 and 4
(d) 2 and 3

The correct answer is (c) 1 and 4.

✅ Correct Answer: (c) 1 and 4

Explanation:

To successfully amplify a DNA sequence using PCR (Polymerase Chain Reaction), the following conditions must be met:

1. Primers must be complementary to the flanking sequences of the target DNA.
2. One primer should bind to the 5′ to 3′ strand of the target DNA, and the other should bind to the 3′ to 5′ strand.
3. DNA synthesis occurs in the 5′ to 3′ direction, so the primers should be oriented toward each other.

Given DNA Sequence:

5’GACCTGTGGAAGC…………..CATACGGGATTGA3′
3’CTGGACACCTTCG……………GTATGCCCTAACT5′

Primer Pair Selection:

1. Primer 1: 5'GACCTGTGGAAGC3' → Matches the 5′ end of the sense strand → Acts as the forward primer.
2. Primer 4: 5'TCAATCCCGTATG3' → Reverse complement of the antisense strand at the 3′ end → Acts as the reverse primer.

Why Other Options Are Incorrect:

How PCR Works with the Correct Primers:

1. Denaturation: Double-stranded DNA is heated to separate the strands.
2. Annealing:
   • Primer 1 binds to the 5′ end of the sense strand.
   • Primer 4 binds to the 3′ end of the antisense strand.
3. Extension: DNA polymerase extends both primers, synthesizing complementary DNA strands.

Primer Orientation and Complementarity:

Applications of PCR-Based Amplification:

✅ 1. Molecular Cloning

• Amplify target genes for insertion into plasmids.

✅ 2. Mutation Analysis

• Detect and analyze genetic mutations.

✅ 3. Pathogen Detection

• Identify bacteria and viruses using gene-specific primers.

✅ 4. Genetic Fingerprinting

• Amplify and analyze variable DNA regions for forensic studies.

Summary:

• The correct primer pair for successful amplification of the DNA sequence is Primer 1 and Primer 4.
• Primer 1 binds to the 5′ end of the sense strand, and Primer 4 binds to the 3′ end of the antisense strand — enabling successful PCR amplification.