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Restriction Enzyme and Sequencing Problems - (Sep/29/2006 )

Okay here is a problem.

I found a mutation in a gene. The wt allele has 2 RE cut sites at the mutation site. I use the REs to
geneotype. Single smaller band is WT, double band is HET, and upper band only is HOM. Pretty simple.

But, just to make sure everything was correct, i decided to sequence the mutated region. To my dismay, the sequencing result was entirely different compared to the RE results.

What is going on? Both sets of results, now that i look at it, is quite confusing in that even the phenotype does not match the genotyping results.

Has anyone had similar experiences?

Many thanks!



On the face of things, the results certainly don't add up. So the problem must lie in the detail.

  1. Are you certain that the restriction sites used in the PCR digest were not found in the sequence of the PCR product?
  2. Did the sequence data come from the PCR product?,ie an aliquote of a PCR sample which you are certain (by digestion) contains either the WT, HET and HOM digestion site types.
I find it very odd that the two do data sets do not match. Could you post the sequence data, (with the appropriate parts highlighted) and a gel photo (with anotation) of the PCR digest. Clues to this puzzle must lie somewhere in the data.