Path: utzoo!utgpu!news-server.csri.toronto.edu!rpi!usc!snorkelwacker.mit.edu!thunder.mcrcim.mcgill.edu!bonnie.concordia.ca!ccu.umanitoba.ca!frist From: frist@ccu.umanitoba.ca Newsgroups: bionet.molbio.methds-reagnts Subject: Why does RAPD mapping work? Message-ID: <1991Mar30.213418.20252@ccu.umanitoba.ca> Date: 30 Mar 91 21:34:18 GMT Organization: University of Manitoba, Winnipeg, Canada Lines: 65 THE TECHNIQUE The latest 'hot' technique making the rounds is RAPD mapping. For those of you not familiar with the technique, it basically involves the use of short PCR primers to amplify genomic DNA, generating genotype-specific patterns of bands, which segregate just as traditional RFLP markers do. For a mapping project, the investigator will typically screen a hundred or more primers of random design, choosing primers that give a manageable number of bands (eg. 5 or 6), and of those primers, choosing as informative ones those that detect polymorphism (ie. presence vs. absence of a band) in the population. Linkage is established as with RFLPs. RAPD (pronounced 'rapid') mapping is described in: Williams, J.G.K et al. (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. NUCL. ACIDS RES. 18:6531-6535. 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) one 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. The authors don't even ATTEMPT to explain why RAPD mapping works. Is there any experimental evidence that will shed some light on this? Or is it that they simply forgot to mention that they restrict their DNA? 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? The only way I can see this working is if the primers represented inverted repeats that were close enough together to allow both strands to amplify. Since the average primer length is 10nt, a template site should occur every 4e10 nt (~10e6nt), which means perhaps a thousand or more sites in a typical eukaryotic genome. But given one site, the probability of a second site occuring is also 4e10, so the average distance between the two sites should be 10e6nt. Since the bands seen by the authors were typically in the 0.5 to 3.0 kb range, we can exclude the idea of a distal perfect 10-mer match in the opposite orientation occurring within this distance. Perhaps the best explanation is that the second site does not have to be a perfect match, but need only match several (eg. 6) nucleotides at the 3' end of the primer. Exactly such a phenomenon appears to occur in 'primer dimer' formation, in which some primers can prime themselves by pairing at their 3'ends, creating two 3'recessed ends. This is well documented. If imperfect 2nd strand priming is the explanation for RAPD mapping, then a suitable downstream site would occur within a few thousand bp. Of equal importance, this mechanism would also predict that BOTH strands would amplify, resulting in exponential, rather than linear growth. Anyone care to comment? =============================================================================== Brian Fristensky | Department of Plant Science | Can you say University of Manitoba | Winnipeg, MB R3T 2N2 CANADA | CHICKEN UBIQUITIN, CHICKEN UBIQUITIN frist@ccu.umanitoba.ca | CHICKEN UBIQUITIN, CHICKEN UBIQUITIN, Office phone: 204-474-6085 | CHICKEN UBIQUITIN, CHICKEN UBIQUITIN FAX: 204-275-5128 | CHICKEN UBIQUITIN, CHICKEN UBIQUITIN...? ===============================================================================