Path: utzoo!utgpu!news-server.csri.toronto.edu!rpi!crdgw1!uunet!av8tr!elliston
From: elliston@av8tr.UUCP (Keith Elliston)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Why does RAPD mapping work?
Summary: one thing not to forget....
Message-ID: <645@av8tr.UUCP>
Date: 2 Apr 91 14:26:12 GMT
References: <1991Mar30.213418.20252@ccu.umanitoba.ca>
Organization: Aviators Online BBS, Somerville, NJ
Lines: 52

In article <1991Mar30.213418.20252@ccu.umanitoba.ca>, frist@ccu.umanitoba.ca writes:
> 
> THE PROBLEM
> Here's my question. At no point in the paper do the authors even hint that
> they have restricted their genomic DNA. Yet, they get reproducible, character-
> istic band patterns for each different primer. Note that only 1 (count 'em) on
> primer is used in each reaction. Theoretically, elongating chains for a given 
> primer should stop within a range of distances from the starting point, rather
> than at discrete sites, UNLESS the DNA has been restricted, in which case all
> chain elongation should stop at the first RE site distal to the primer.
.
.
> 
> Another problem: If RAPD mapping is simply doing a linear amplification, 
> rather than a true polymerase chain reaction, which is exponential, I am a 
> little skeptical that you would see bands in EtBr staining, even after 45
> cycles (ie. making 45 copies of the starting material)
> 
> THE SOLUTION?

Brian explains PCR and the techniques of RAPD pretty well.  In fact, this was
the first I have heard of them, but then again, I don't do much in the lab
anymore.  But, there is one thing that many people using PCR routinely forget.
Once you have amplified the first fragment from the genomic DNA, the sequence
of the PCR primer is now EXACTLY represented in your copied fragment.  So, in
the case of the RAPD technique, as long as you get one discreet fragment from
the first 2 rounds (so that the primer sequence is EXACTLY represented in
the fragment, at both ends) the suceeding rounds will amplify THAT FRAGMENT
very very well.  So, you may get some mismatch priming in the first 2 rounds
of synthesis, but after that, you will be hybridizing the PCR primers to 
exact copies of themselves, not the mismatched sequence in the genome.

One way I have seen PCR misused in this way, is in PCR sequencing.  People
tend to forget that they NEVER see the actual starting sequence that is 
under the PCR primers.  That sequence is always copied from the primers
themselves, and does not represent that sequence found in that position
in the original DNA.  Remember that you are EXTENDING the PCR primers,
and that the second round of synthesis uses that site for the hybridization
of the primers in suceeding rounds of synthesis.

I am not an expert in the field, but this is my thinking on the situation.

Later,

Keith 


-- 
Keith O. Elliston          elliston@av8tr.UUCP           elliston@msdrl.com
AA5A N9734U                elliston@mbcl.rutgers.edu     elliston@biovax.bitnet
"Beware of pseudo-experts with a mission and a grudge, especially if they are
 lawyers pretending to be scientists." -- H.W. Lewis in 'Technological Risk'