RFLP/AFLP, LFRFLP... - description of techniques? (Jan/14/2006 )

Hi,
I would really appreciate if anyone would tell me about the main differences between AFLP and RFLP and give me some example of their use. I am also interested about when LFRFLP (low frequency RFLP) is used. If possible, examples from microbial ecology would help me most.
Thanks in advance!

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Hello,

RFLP: Restriction fragment length polymorphisms. DNA is cut with informative RE's and separated on a gel and for length differences of the fragments is looked for (i.e. due to a mutation leading to a loss of a cutting site, leading to a longer fragment). Due to the huge number of cutting sites, hybridization and radiolabeling may be necessary or better PCR-RFLPs, were PCR amplified fragments are cut. RFLps are codominant markers and can be analyzed similar to alloenzymes.

AFLP: Amplified fragment length polymorphisms
Modified RFLP method. Genomic DNA is restricted using a common site- and a rare site-cutter (e.g. MseI and EcoRI, respectively) "Adapter" oligonucleotide sequences are ligated onto these DNA fragments. (AFLP adapters consist of two parts, a core sequence and an enzyme specific sequence). A radioactive (32P) PCR reaction, made stringent using high annealing temperatures, is then used with primers consisting of three parts: an enzyme specific sequence specific to the
adapter-sequences; a selective extension determining the specificity of the adapter to the enzyme by 1–3 arbitrary nucleotides on the 23’ end of the primer and finally, the CORE sequence of the primer itself. Polymorphisms are amplified and visualized on polyacrylamide sequencing gel
using autoradiography. Therefore it is a quite complicated method using DNA of unknown or random sequence, respectively (but needs no cloning and sequencing). This only allows to use it as dominant marker.
Hope this helps.
P.S: I have several more paper about the general theme "molecular markers and there use" if you're interested, but I must search a little bit.

regards
P.

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Thanks, a lot..you've been very helpful. So the main difference between PCR-RFLP and AFLP is, that in the first case you amplificate a fragment (f.e. ribosomic genes) and than make a restriction analysis, but in the second case you make restriction analyisis (possible also to do the hole bacterial genome?) and than amplify the fragments? Sorry if the question is a bit stupid, but I am really in a hurry (studying for exam this week), so don't have time to read some articles by my self...
THANKS AGAIN!

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Hi, sorry for late response.
What you stated is correct, but not the point. RFLP you can also do without PCR (eg using RE's on total genomic DNA = you would get a smear on the gel, so you hybridize the cut DNA on membran with known DNA of interest and then make autoradiographies. PCR-RFLP is the faster and easer way...But normally you work on only some loci because its to time-consuming to use many enzymes and many different DNA loci ergo: so not so high resolution (you detect less variability) but this are codominant marker, which is for data analysis better.
AFLP is for for genotyping individuals for a large number of loci using a minimal number of PCR reactions: The three steps are:
1. DNA is cut with restriction enzymes (e.g. MseI and EcoRI, see above) and then linkers are ligated on.
2. Pre-selective PCR is performed using primers which match the linkers. These primers have a two base overhang. Each combination (two bases on the MseI linker and two bases on the EcoRI linker) reduces the number of DNA fragments by 256.
3. Selective PCR is performed using primers with three base overhangs. For any given pre-selective amplification, there are 16 possible selective primer combinations that can be used. The EcoRI primer is labeled so that only fragments that contain an EcoRI site will be detected.
So you have much higher resolution, but you use random DNA and have dominant markers.
Hope its not to late
P.

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Thanks a lot, really! Now I got a better picture!
Kind regards!

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