I have seen this effect when the 3' end of one primer sets over a polymorhism ( not the one you are looking for). Then under pcr annealing conditins both alleles amplify but under the different annealing temperature of the sequencing reaction one allele does not amplify so the mutant allele is either very weak or non existant

Thank u for the fast reaction...actually not amplification of one of the allele due to not suitable annealing may explain this phenomenon...but still it is strange because the same two forward primers also could detect mutation in one particular patient...so the problematic Forward primer sometimes detect,sometimes-not the mutation..It is easy to discard the problematic primer,but I am.curious what the reason behind this issue is?

Thank u once again

possibly your patient does not have the misfit polyorphism so bot alleles amplify and sequence. \it is worth checking dbsnp database for rare snps in your region of amplification. If the polymorphism under the primer is not linked to the snp you are sequencing then it is quite random whether the primer binds to the snp containing or normal allele

1. Primer Binding Specificity

Primers are short, single-stranded DNA sequences that bind to complementary sequences on the template DNA. The specificity of this binding is crucial for successful PCR amplification. A single nucleotide difference (mutation) in the primer binding site can significantly affect the binding affinity and specificity.

Factors Influencing Primer Binding Specificity:

• Melting Temperature (Tm): The Tm of a primer is the temperature at which half of the primer-DNA duplex is dissociated. A single nucleotide mismatch can lower the Tm, reducing the stability of the primer-template complex.
• Binding Energy: The binding energy between the primer and the template is influenced by the number and type of hydrogen bonds formed. A single nucleotide mismatch can disrupt these bonds, reducing the overall binding energy and making the primer less effective.
• Primer Length: Longer primers generally have higher specificity because they form more hydrogen bonds with the template. However, a single mismatch in a long primer may still be tolerated if the overall binding energy is high.

2. Primer Design and Sequence Context

The sequence context surrounding the mutation can also influence primer binding. Even if two primers are almost identical, a single nucleotide difference at a critical position can affect their ability to detect the mutation.

Key Considerations:

• 3' End of the Primer: The 3' end of the primer is particularly important for initiating DNA synthesis. A mismatch at the 3' end can prevent the primer from binding effectively and initiating PCR amplification.
• Secondary Structure: The secondary structure of the primer and the template can affect binding. A single nucleotide change can alter the secondary structure, making it more difficult for the primer to bind.

3. Mutation Type and Position

The type and position of the mutation within the primer binding site can also influence detection.

Types of Mutations:

• Transition vs. Transversion: Transition mutations (purine to purine or pyrimidine to pyrimidine) are more common and may be less disruptive to primer binding than transversion mutations (purine to pyrimidine or vice versa).
• Position of the Mutation: A mutation near the 3' end of the primer is more likely to disrupt binding than a mutation near the 5' end.

4. PCR Conditions

The PCR conditions, such as annealing temperature and magnesium ion concentration, can also influence primer binding and amplification efficiency.

Optimization of PCR Conditions:

• Annealing Temperature: The annealing temperature should be optimized to ensure that the primer with the correct sequence binds preferentially. A lower annealing temperature may allow non-specific binding, while a higher temperature may prevent the primer from binding at all.
• Magnesium Ion Concentration: The concentration of magnesium ions (Mg2+) in the PCR reaction can affect the stability of the primer-template complex. Optimizing the Mg2+ concentration can help improve specificity.

Example Scenario

Consider two forward primers, Primer A and Primer B, that are almost identical except for a single nucleotide difference at the 3' end:

• Primer A: 5'-ATCGATCGATCGATCG-3'
• Primer B: 5'-ATCGATCGATCGATCA-3'

If the mutation in the genomic DNA changes the last nucleotide of the primer binding site from G to A, Primer A will not bind effectively because of the mismatch at the 3' end. In contrast, Primer B will bind effectively because it has an A at the 3' end, matching the mutated sequence.