Register | Sign In


Understanding through Discussion


EvC Forum active members: 65 (9162 total)
7 online now:
Newest Member: popoi
Post Volume: Total: 915,814 Year: 3,071/9,624 Month: 916/1,588 Week: 99/223 Day: 10/17 Hour: 6/1


Thread  Details

Email This Thread
Newer Topic | Older Topic
  
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 6 (360160)
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

Replies to this message:
 Message 3 by EZscience, posted 10-31-2006 4:17 PM crashfrog has not replied
 Message 4 by Wounded King, posted 10-31-2006 5:51 PM crashfrog has not replied
 Message 5 by kuresu, posted 10-31-2006 8:05 PM crashfrog has not replied
 Message 6 by U can call me Cookie, posted 11-01-2006 8:13 AM crashfrog has not replied

  
AdminJar
Inactive Member


Message 2 of 6 (360167)
10-31-2006 2:43 PM


Thread moved here from the Proposed New Topics forum.

  
EZscience
Member (Idle past 5153 days)
Posts: 961
From: A wheatfield in Kansas
Joined: 04-14-2005


Message 3 of 6 (360182)
10-31-2006 4:17 PM
Reply to: Message 1 by crashfrog
10-31-2006 2:32 PM


Well I'm not a geneticist, but it seems to me that the purpose of PCR is to usually to make a large number of copies of one specific sequence to use for other purposes, purpose for which many, many copies of a particular sequence are needed.
On another note, if RLFP using the selected enzyme actually produces a series of fragments that corresponds to the profile of different fragment sizes predicted by the sequence, this would provide some independent confirmation that the sequence has in fact been correctly inferred and cleaves as anticipated. No such confirmation would be obtained from a simulation.
But I'm just speculating here until one of the gel-jockeys shows up...

This message is a reply to:
 Message 1 by crashfrog, posted 10-31-2006 2:32 PM crashfrog has not replied

  
Wounded King
Member
Posts: 4149
From: Cincinnati, Ohio, USA
Joined: 04-09-2003


Message 4 of 6 (360208)
10-31-2006 5:51 PM
Reply to: Message 1 by crashfrog
10-31-2006 2:32 PM


One major factor would be why is she doing it? Is it just because all the sequences are there? Do the different species need discriminated?
If for instance you were trying to produce an assay to discriminate between different organisms based on this particular sequence then an in silice approach would plainly be useless unless you re-sequenced your product every time.
As far as simple phylogenetics of the sequences already gained goes I don't see any point to doing subsequent RFLP. But if you wanted to analyse further samples then having an already optimised set of enzymes which could distinguish distinct species or haplotypes could be useful. On a southern blot you could simultaneously analyse multiple samples and have actual bands on gels to show for it, simply cloning and sequencing every sample would be less satisfying, probably as expensive and time consuming (though probably not for your wife herself) and would allow the introduction of a further layer of possible experimental error in the case that the sequencing reads were off, you might have to sequence every PCR product multiple times to be certain.
This isn't really my field but these are a few possibilities that come to mind.
TTFN,
WK

This message is a reply to:
 Message 1 by crashfrog, posted 10-31-2006 2:32 PM crashfrog has not replied

  
kuresu
Member (Idle past 2513 days)
Posts: 2544
From: boulder, colorado
Joined: 03-24-2006


Message 5 of 6 (360249)
10-31-2006 8:05 PM
Reply to: Message 1 by crashfrog
10-31-2006 2:32 PM


I might have an answer for you on thursday, maybe next tuesday. the lab I work for (for my work-study) is involved in a lot of DNA stuff, and this sounds real similar to what they're doing. Me, I just work for the enzyme analysis section.
(oh, as to the project, the lab, headed by Steve Schmidt*, is analyzing the effect of apline fungal species on the biome . . .or something to that effect)
*not on the project steve list, unfortunately

Want to help give back to the world community? Did you know that your computer can help? Join the newest TeamEvC Climate Modelling to help improve climate predictions for a better tomorrow.

This message is a reply to:
 Message 1 by crashfrog, posted 10-31-2006 2:32 PM crashfrog has not replied

  
U can call me Cookie
Member (Idle past 4953 days)
Posts: 228
From: jo'burg, RSA
Joined: 11-15-2005


Message 6 of 6 (360349)
11-01-2006 8:13 AM
Reply to: Message 1 by crashfrog
10-31-2006 2:32 PM


The whole point of an RFLP assay is to cheaply check the allele state of a Single Nucleotide Polymorphism (SNP).
If your wife sends all her samples to be sequenced, then this totally negates the need for an RFLP system (real or simulated), since all you'd have to do to assess the allele state of the SNP is read the sequence trace.
Whether or not one would simply sequence all one's samples depends on how long a length of DNA one is working with, and how much funding you have to play with.
If your wife is working on the D-loop of mtDNA only, then sequencing is the way to go, since lots of SNPs in relatively short sequence.
However, its high rate of mutation can be problematic in phylogenetic studies.
Nowadays, people are also doing mtDNA whole genome sequencing; way better resolution; however the cost of this is still substantial, especially when compared to RFLP analysis.
For this reason, its quite common that you find people sequencing the D-loop and using a few RFLPs in conjunction to this in order to better resolve certain haplogroups (this is from the perspective of someone who works with human samples).
If your wife is working with a relatively large number of SNPs located outside of the D-loop; and she has access to a capillary sequencer, she might consider using Single Base Extension (SBE). Using this, one can multiplex SNPs, which would save a substantial amount of time and effort. Its also relatively cheap.
Typing of mitochondrial DNA coding region SNPs of forensic and anthropological interest using SNaPshot minisequencing

"The good Christian should beware the mathematician and all those who make empty prophecies. The danger already exists that the mathematicians have made a covenant with the devil to darken the spirit and to confine man in the bonds of hell." - St. Augustine

This message is a reply to:
 Message 1 by crashfrog, posted 10-31-2006 2:32 PM crashfrog has not replied

  
Newer Topic | Older Topic
Jump to:


Copyright 2001-2023 by EvC Forum, All Rights Reserved

™ Version 4.2
Innovative software from Qwixotic © 2024