PURPOSE:

To apply Restriction Fragment Length Polymorphism technique to identification of person.

INTRODUCTION:

What's a RFLP?

RFLP stands for Restriction Fragment Length Polymorphism. The name is a bit imprecise, but here's what it means.

Basically, a RFLP is a band in a gel (or in a southern blot produced from a gel). So why not just call it a "band" and leave it at that? Because of the way in which these bands are used for genetics.

Here's an example. Let's suppose that we have an RNA probe that binds to a unique region in the a-globin gene:

If we do an experiment in which we isolate total human DNA (genome) , cut it with a restriction enzyme (say, EcoR1), separate the millions of restriction fragments on a gel, and then do a Southern blot using the RNA probe, we might find a single band in the blot. We can treat that band almost as if it were an inherited trait, like blue eyes, black hair, or hemophilia, because it is inherited, being a small piece of DNA.

Let's suppose that we took DNA samples from 1,000 individuals, and prepared the same Southern blots for each of them. If we found that every one of them had a band at exactly the same position, we would say that this particular restriction fragment is "monomorphic" in the population, meaning that only 1 form of it is found. Monomorphic restriction fragments aren't usually interesting for geneticists. If everybody has the same version of a particular trait, you can't do very much genetics with it. (A probe taken from the middle of an important gene, like the globin gene, is most likely to identify a monomorphic fragment, and is therefore uninteresting for genetics or individual identification)

Nowadays, we can use PCR thermocycler in order to amplify certain polimorphic sides, and we can add several restriction enzyme to our PCR product, and run them on agarose gel and chack the bands under UV light. By this method we save our both money and time.

However, let's suppose we found several different bands in the population, as shown in this Southern blot of samples from 6 individuals:

These restriction fragments would be said to be "polymorphic" in the population, since there are several different forms of them. We would have discovered a Restriction Fragment Length Polymorphism, or RFLP

From now on we'd point to samples 1, 3, and 5 and say that they contained the same RFLP, that sample 2 had a smaller RFLP, sample 4 a larger one, and that sample 6 had two RFLPs, all using the same probe. The bands are properly referred to as "RFLPs," and they represent differences in DNA organization between individuals.

What do RFLPs "do" in the cell?

Well, remember that a RFLPs doesn't "exist" in nature any more than a "photograph" exists without a photographer to make it. A RFLP is something that we make from the genome, not something that exists on its own. Therefore, some RFLPs are produced from DNA sequences in genes (both introns and exons), some from controlling regions like promoters, and many from the bulk of DNA, which seems to have no function at all. In fact, most RFLPs used in criminal work have no function at all, but, like other RFLPs, they can be used by an investigator to identifiy individual DNA, to map genes or to follow their passage from one generation to the next. We can use this method for; detecting stolen animal, losted baby, cemetery place and indentify person.

A palindrome is a word, phrase, number or any other sequence of units (like a strand of DNA) which has the property of reading the same in either direction two fold rotational symmetry, it can be rotate 180 degree without change in base sequence.

3’---CTTAAG---5’

5’---GAATTC---3’

A haplotype is a combination of genotypes on the same chromosome that tend to be inherited as a group. In other words, it is the genotype for a group of genes.

With this in mind, let's modify the definition of haplotype to: the observed patterns (various combinations) of alleles for the genes in a particular gene cluster.

Certain patterns of alleles in gene clusters tend to be preserved and inherited as a group because, in general, the closer genes are together on a chromosome, the less likely they are to recombine.

MATERIAL:

METHOD:

We take sample kits contains 8µl amplified DNA and add 1µl buffer and 1µl enzyme mix (BamH1 & Hind3 restriction enzymes). We incubate at 37C0 for few hours. After incubation we add 3µl bromephenol blue and impulse centrifugate them. Finally, we load them on agarose gel and run at 150V for 15 – 20 minutes. We observe the bands on the gel under UV light and decide which sample can be murder.

RESULT:

DISCUSSION:

We observed crime sample & sample #4 give exactly same bands on the same bp lenght, so that #4 sample can be murder by carrying identicaly same type polimorphic regions. Other samples not contain that specific restriction sides on teher polymorfic region. #5 also carries one band identically with crime scene, but it’s second band so smaller size than crime scene second band.

We use bromephenol blue in order to dye DNA samples and easily load on the gel and also increase the weight of DNA, so DNA easily drop buttem of the gel. We must use equal volume of specific enzyme’s buffer to prevent enzyme de activation with high temprature, charge currency ,and pH changes.

 

KAYNAKLAR: