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Author Topic:   Question for Geneticists: Simulating RFLP
crashfrog
Member (Idle past 1466 days)
Posts: 19762
From: Silver Spring, MD
Joined: 03-20-2003


Message 1 of 2 (360159)
10-31-2006 2:32 PM


I have a question for the molecular geneticists, prompted by a conversation with my wife last night. She's doing some phylogenetics work (I think I've mentioned this before) and as part of that she sent off some PCR (polymerase chain reaction) results to a lab to be sequenced (presumably via dye-termination method.)
When they come back, she has essentially a text file with the nucleotide sequence of her PCR products, along with the dye-termination results. It looks something like this:
So, essentially, she has a literal readout of the base pair sequences for a given stretch of mitochondrial DNA.
What's weird to me is what she uses this information for. One of the tools she uses is Webcutter, which is a tool used to choose restriction enzymes for PCR-RFLP (restriction fragment length polymorphism) analysis; based on her sequences she can see where various simulated commercially-avaliable restriction enyzmes will clip her various sequences, helping her choose which enzyme to use for RFLP. When she finds one that looks like it's going to successfully discriminate between the different species she has samples for, she prepares the actual PCR-RFLP, waits a few hours, and then runs a laborious set of gel electroporesis runs to get results, which usually takes her weeks to complete with a graduate student's schedule.
What I don't understand is why RFLP is even needed once you have the nucleotide sequences. Cleaving text strings according to patterns is a trivial programming task (as evidenced by the Webcutter), whereas RFLP, while not expensive, is complicated, hazardous, and time-consuming. And not always satisfactory - my wife found an almost perfect restriction enzyme to use, but discovered that she couldn't use it because it would be impossible to successfully read the resulting gel electroporesis run. Whereas, simulating the restriction analysis would have made the gel's readability problems moot since you could simply directly inspect the length of the resulting restriction fragments.
None of her professors or advisors have supplied a reason why merely simulating RFLP is insufficient, and I think she's afraid to ask for fear of looking stupid in her department. I, on the other hand, have nothing to lose, so I thought I would ask. In an age when direct gene sequencing is cheap and easy, why do we still have to do RFLP in the lab when we could easily do it on the computer? What does the real RFLP tell us that a simulated RFLP would not?
Edited by AdminJar, : fix display width of image

AdminJar
Inactive Member


Message 2 of 2 (360166)
10-31-2006 2:43 PM


Thread copied to the Question for Geneticists: Simulating RFLP thread in the Is It Science? forum, this copy of the thread has been closed.

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