Re: How does DNA fingerprinting work with respect to separating the alleles?

Hello,

Thanks for your interesting question. I think the best way to answer it is to approach it is to ignore the information in the various genome mapping projects for the moment, (we'll come back to that later), and think about what things were like before we had such detailed information about the sequences of some organisms.

So, you have DNA, and what you do know is that it over the majority of a genome, there will be some differences between individuals. Restriction enzymes recognize short sequences within DNA, and at any point where a difference between individuals occurs in the cleavage site of a restriction enzyme, an RFLP exists.

DNA samples can be cut with a particular restriction enzyme and run down a gel according to size. The contents of the gel can then be transferred onto a membrane (a Southern blot). This membrane now contains a long "smear" of DNA, with big fragments at the top and smaller and smaller fragments present as you go down.

To detect the RFLP, you need to have a unique probe (which is complementary to a region of the DNA samples). The probe is labelled in some way, (e.g. with radiation or fluorescence), and is then hybridised with the DNA on the Southern blot. The probe should bind the region of the DNA on the membrane complementary to it. If this region of the DNA has been cut with the restriction enzyme in one sample, but not another, you have found an RFLP. This would show up on the blot as one sample having a single band visible, and the other having two bands. A diagram of this can be found at:

NCBI

Most RFLP analysis took place before we had sequence data for the regions of interest, so the answer to your question is that the restriction enzymes used are not (could not have been) specifically chosen beforehand to cut at a site that produces the RFLP. The way a restriction enzyme would normally be chosen is to take a panel of DNA samples and cut them with various restriction enzymes, (i.e. for one test, the samples are all cut with a particular enzyme, for a second test, the whole panel are cut with a different enzyme, and so on). Each set of DNA samples cut with a particular enzyme could then be blotted, and a probe applied to them. Any restriction enzyme that allowed the identification of an RFLP with respect to the probe used is noted. That restriction enzyme, in conjunction with that particular probe, can be used to detect different alleles.

There is a good chance of finding RFLP's using a method like this since they are a fairly common type of variation in nature.

DNA fingerprinting can be carried in other ways apart from using RFLP's, and in fact, other methods are faster and more convenient. For example, one can use randomly amplified polymorphic DNA (RAPD), which is generated using PCR technology. A single PCR primer designed at random will often, by chance, amplify several different regions of the genome. The result is a set of different-sized amplified bands of DNA. These bands may be different sizes in different individuals, and may be used as molecular markers (heterozygous loci). A diagram of this process is available at: NCBI

Some other polymorphic markers in use include:

-VNTR's (variable number of tandem repeat units). These consist of tandem repeats of at least 16 nucleotides where the number of repeats may vary between individuals.

-microsatellites, which are repeat sequences consisting of 2, 3, or 4 nucleotides. These are common in most organisms, and individuals may differ in the number of repeats at a locus.

-SNP's -pronounced "snips" (single nucleotide polymorphisms). These are differences in just one nucleotide between individuals. Often the SNP sites found are bi-allelic, (that is, only 2 alleles are present in a population). The sequence of a region needs to be known to identify this type of polymorphism. SNP's are useful in studying population genetics, and may have a role in identifying disease related genes. Their use is often suggested in the possible future development of customised (perhaps even personalised?) drugs.

You can read more about SNP's at: http://snp.cshl.org/about/

If you want to see some SNP's, try: http://snp.cshl.org/db/snp/map

I seem to have moved off the topic of RFLP's! If you want to know more about them, others have already done a great job of writing about them. To make things even better, recently, the National Centre for Biological Investigations (NCBI) in the USA have made access to some science text books free. You can go to this site to see what is available: http://www.ncbi .nlm.nih.gov:80/entrez/query.fcgi?db=Books

The books "An Introduction to Genetic Analysis" by Griffiths et. al, and "Modern Genetic Analysis" by Griffiths et al, both available through this site, contain a good amount of information on RFLP's, and probably lots of other information you might find of use in your teaching.

It may be useful for you to remind your students that the large amount of genomic sequence data we currently have for some (by no means all!) organisms is a new development. Most of the work carried out in years past was done without knowing the exact sequence of the regions of DNA of interest. The experimental designs employed reflect the amount of knowledge during the period they were developed, and keeping this in mind may help in understanding some of the techniques of molecular biology.

To finish off this email, I'd like to make a subjective comment about the wording of your question:

"What I am wondering is if restriction enzymes used in DNA fingerprinting are specially chosen so as to cleave the chromosome with respect to the different alleles that it contains?"

While you are correct that the restriction enzymes are cleaving the chromosome, I have not really heard it worded that way, unless one was discussing infrequent restriction enzyme cutters (e.g. Not1), which would still leave the DNA in fairly large chunks which could then be separated by size. (If the organism being studied has small enough chromosomes, you can separate them using an electric current, without actually cutting them at all.) With RFLP's, the DNA is usually getting chopped into many, many much smaller pieces, and so it is probably more common to refer to the "DNA" being cleaved, rather than the "chromosome".

Also, it is not that common to talk about a chromosome "containing" alleles. It is more common to talk about a gene being located on a chromosome, and there being alleles of that gene.

I hope that somewhere in all this I have answered the question you were asking. Please get back in touch if I have not. The terminology of this area of biology is a challenge at first, but I'm sure that if you refer to some of the recent genetics textbooks out there, that it will all fall into place.

Good luck!

1 Schleif, R. Genetics and Molecular Biology. The John Hopkins University Press. Baltimore. 1993. ISBN: 0-8018-4674-9