frist@ccu.umanitoba.ca (03/31/91)
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...? ===============================================================================
elliston@av8tr.UUCP (Keith Elliston) (04/02/91)
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'
cmslanigan@ucdavis.edu (Caroline Lanigan) (04/06/91)
In article <1991Mar30.213418.20252@ccu.umanitoba.ca> frist@ccu.umanitoba.ca writes: >... 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...Exactly such a phenomenon appears to occur in 'primer >dimer' formation, .... > >Anyone care to comment? > >=============================================================================== >Brian Fristensky, Department of Plant Science, University of Manitoba >Winnipeg, MB R3T 2N2 CANADA, frist@ccu.umanitoba.ca >Office phone: 204-474-6085 FAX: 204-275-5128 >=============================================================================== > Hi Brian, I have been working with RAPD PCR since Sept and can comment on some of your concerns: 1. The DNA is not restricted. 'Whole" cellular DNA, within the limitations of the isolation technique, is used in the reaction. And yes, consistent, reproducible banding patterns are observed on EtBr stained, agarose gels. PCR amplification of sequences up to 10 kb have been reported and longer amplifications have been implied (see Cetus literature). Ifthe DNA was restricted and amplification occurred from a single priming site, the amplification product would be single-stranded. From my experience this is not the case: I observe complex, repeatable, banding patterns of double stranded DNA in EtBr stained agarose gels. In fact, if the DNA were restricted and amplification occurred from a single site per fragment, no product would be observed on the gel because no subsequent priming would occur. The fact that easily observable bands occur supports exponential amplificaton of primed sites. I routinely use an arbitrary 10 mer primer for each reaction, where the G+C:A+T content is 60:40. In theory, this single 10 mer sequence occurs as multicopy DNA throughout the genome, in both orientations. This is not unexpected, given the nature of genomic organisation in living systems (recall highly repetitive, moderately repetitive, and unique DNA from Cot analysis) and the fact that the sequence arrangement of much of this DNA is unknown (with the exception of sequences like Alu and transposable elements like Ac/Ds). 2. Not every primer works. The non-random organization of genomes of living organisms is well known and supports this fact. My primers are randomly generated within the caveat of being composed 60% G+C. The observation of 5 to perhaps 50 bands, instead of the 500 according to your calculations, is also explained by the nonrandom nature of genomic DNA organization. 3. The notion that one or both of the primers are mismatched at hybridisation is not supported by the reaction conditions. Although the annealling temperature of the reaction appears low, at 35 degrees C, in fact this is a very stringent reaction. I calculated the Tm for my primers and found it to be 30 degrees. I do not believe that even a single base mismatch could occur under these conditions, effectively ruling out any mismatching of the primers. If mismatches were possible, the reaction would not be reproducible between experiments. Mismatching allows for more different priming locations which would not be the same between reactions. In my experience, the banding patterns of amplification products are reproducible, within certain caveats. Note that primer-dimers occur when the ends of primers are complementary, not when they are identical. If this doesn't convince you that RAPD PCR works, I'd be happy to continue this discussion, or watch for my paper on RAPD PCR in monkeys (in preparation) to be out hopefully this summer! P.S. Is Dan Gietz still there? He is/was on the faculty in the Dept of Genetics, Biology or perhaps medicine? Caroline Lanigan, Comparative Genetics, California Regional Primate Research Center, University of California, Davis CA USA 95616