SNAPSHOT PCR QUERY

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Firoz Ahmad
Firoz Ahmad's picture
SNAPSHOT PCR QUERY

Hello everyone,
This is regarding MTHFR C677T mutation detection by Single Nucleotide Primer Extension assay. My snapshot primer is 34 mer and when I did a Primer Focus Reaction I got C at 34.5 and T/A/G in the range of 43-44. I did about 10 clinical specimens and have got all the wild type specimens i.e. C at 34.5 and mutants i.e. T at 43.4. All the results were confirmed by sequencing. I fail to understand how a primer that is 34 mer shows mutant peak at 43.4 (almost 9 bp away) in the electropherogram. My exposure to snapshot assays is minimal. I am very interested to hear the opinions of others regarding this.
Best regards,

Firoz