Warfarin Drug dosage based on two genes - VKORC1 & CYP2C9 (Jul/06/2010 )

Dear all professionals,
Im currently an summer student at a research facility, and I'm given a tiny project that deals with the prospective study of warfarin dosage requirements based on CYP2C9 and VKORC1 genotypes. Sequencing is not a problem, but now that I have to try PCR-RFLP as well, I dont quite get how the primers identify the part of genome sequence that I want. I have two restriction enzymes, KpnI for CYP2C9, and MspI for VKORC1. All I need to know is how these restriction enzymes find the sequence I want and "cut" it.
Thanks a lot, and if I'm in the wrong place of the forum please let me know.

OK... You know how PCR works don't you? Basically (well this is an assumption on my part, iv'e never heard of PCR-RFLP, but I do know both by themselves) PCR-RFLP will be an amplification of the sequence, followed by a restriction digest of the amplified sequence that "should" give you different size fragments depending on the mutation in the amplified sequence. This may mean that you have a combination of different fragments for different mutations such as 1 band (no cut) if no mutation, 2 bands with one type of mutation (1 cut site) and 3 with another mutation (2 cut sites).

As for the enzymes finding the sequence - it is just like any other chemical reaction really, there is stuff floating around in solution and by random chance the two components interact. If the sequence is wrong for the restriction enzyme, it won't cut and will try again later in another place. This is why incubations are generally quite long so as to give the reactions time to take place.

Talk to your supervisor or other lab members about this - they will be able to help, you shouldn't be expected to know everything when you first join a lab.

BTW, if a moderater thinks this is in the wrong place, they will move it, you don't need to multiple post. I personally think this could have gone in the molecular biology subforum.