Degenerated Primers for Amplifying Mouse Gene from Drosophila Sequence

To amplify the mouse version of a gene whose sequence has been reported for Drosophila but not for mouse the primers to be used in PCR should be
(a) Specific forward and reverse primers
(b) Nested primers
(c) Degenerate primers
(d) None of the above

The correct answer is (c) Degenerate primers.

✅ Correct Answer: (c) Degenerate primers

Explanation

If the gene sequence is known for Drosophila but not for mouse, using degenerate primers is the most effective approach because:

🔬 1. What Are Degenerate Primers?

• Degenerate primers are a mixture of oligonucleotide sequences that contain variability at certain nucleotide positions.
• They are designed based on the conserved regions of a gene from one species (e.g., Drosophila) to target the homologous gene in another species (e.g., mouse).
• Degeneracy accounts for the natural variation in codon usage among different organisms.

🔎 2. Why Degenerate Primers Are Suitable

✅ The genetic code is redundant — multiple codons can encode the same amino acid.
✅ If the sequence is not fully conserved between species, degenerate primers increase the chance of successful amplification.
✅ Degenerate primers target the conserved amino acid sequences while allowing for minor nucleotide variations.

🌟 3. Example of Degenerate Primer Design

• If the conserved amino acid sequence is “ATG GCC CAG” (Methionine-Alanine-Glutamine):
  • Possible codons:
    • ATG → Methionine
    • GCC → Alanine
    • CAG → Glutamine
• Degenerate primer example:
  • 5′ – ATG GCN CAR – 3′
    • N = A/T/G/C
    • R = A/G

Why Other Options Are Incorrect

How Degenerate Primers Work in PCR

1. Template Selection:

   • A known sequence from Drosophila is used to identify conserved regions.

2. Primer Design:

   • Design primers with degenerate bases at positions with variability.

3. PCR Reaction:

   • Degenerate primers bind to multiple similar sequences due to base flexibility.

4. Amplification:

   • The PCR process selectively amplifies the mouse gene segment homologous to the Drosophila sequence.

Steps to Design Degenerate Primers for Cross-Species Amplification

✅ 1. Identify Conserved Regions:

• Use bioinformatics tools to align homologous sequences from different species.
• Focus on regions with minimal sequence variation.

✅ 2. Introduce Degeneracy:

• Include degenerate bases at codon positions where variation is common.
• Example: N = A/T/G/C, R = A/G, Y = C/T.

✅ 3. Optimize PCR Conditions:

• Lower annealing temperature to account for primer mismatch.
• Use a gradient PCR to find the optimal annealing temperature.

✅ 4. Use a High-Fidelity Polymerase:

• High-fidelity polymerases like Pfu minimize random errors.

Challenges in Using Degenerate Primers

🔴 1. Reduced Specificity:

• Degeneracy can lead to non-specific amplification.
• Solution: Use touchdown PCR to increase specificity.

🔴 2. Low Efficiency:

• The presence of multiple primer sequences reduces binding efficiency.
• Solution: Increase template concentration and cycle number.

🔴 3. Primer-Dimer Formation:

• Overlapping or self-complementary primers can form dimers.
• Solution: Use software to predict and avoid dimer formation.

Applications of Degenerate Primers

✅ 1. Cross-Species Gene Cloning

• Amplification of conserved genes from different organisms.

✅ 2. Evolutionary Studies

• Studying homologous gene evolution across species.

✅ 3. Pathogen Identification

• Amplifying conserved sequences in viral and bacterial genomes.

✅ 4. Mutagenesis and Functional Studies

• Generating mutants for functional gene analysis.

Example of Degenerate Primer Use

Summary

• Degenerate primers are ideal for amplifying a gene from a related species when the sequence is unknown.
• They increase the chances of successful PCR by accounting for sequence variation due to codon redundancy.
• Proper primer design, optimized PCR conditions, and high-fidelity polymerases improve the success rate.