7. DNA Fingerprinting - Background

Teacher Background
The crux of this activity is the creation of a DNA sequence on a strip of adding tape, replication of the DNA, then using these DNA sequences to perform DNA fingerprinting.

DNA replicationDNA replicationThe base pairing rules help explain the process of DNA replication – how a cell makes an exact copy each strand of DNA just before it divides. First, an enzyme called helicase unzips the DNA down the middle of the ladder, in between the base pairs. Next, an enzyme called DNA polymerase reads one half of the strand, identifies a matching nucleotide, and builds a new partner strand. The process is complicated by the fact that DNA polymerase can only work in one direction along the sugar-phosphate backbone (remember, that the 2 backbones are oriented in opposite directions to one another). Thus, while DNA polymerase can easily run continuously along one strand, known as the “leading” strand, the other “lagging strand” must be assembled in a piecemeal fashion, one section at a time.

DNA fingerprinting is a technique used to distinguish between individuals of the same species using only samples of their DNA. Its invention by Sir Alec Jeffreys at the University of Leicester was announced in 1985.

DNA fingerprinting is used in forensic science, to match suspects to samples of blood, hair, saliva or semen. It has also led to several exonerations of formerly convicted suspects. It is also used in such applications as studying populations of wild animals, paternity testing, identifying dead bodies, and establishing the province or composition of foods. It has also been used to generate hypotheses on the pattern of the human diaspora in prehistoric times.

Two humans will have the vast majority of their DNA sequence in common. The traditional method of DNA fingerprinting uses restriction enzymes that cuts DNA at a specific sequence. Restriction enzymes work by recognizing a specific DNA sequence and cutting the DNA within this sequence. For instance, the restriction enzyme known as Sma1 looks for the sequence CCCGGG and cuts the DNA between the middle C and G. Other restriction enzymes make a staggered cut with some overhang on each end. For instance, the restriction enzyme EcoR1 looks for the sequence GAATTC and makes a staggered cut leaving what is known as a sticky end.

If designed correctly, a restriction enzyme can target a part of the genome that his highly variable from person to person such that some individuals’ DNA will be cut while others won’t, resulting in variable length DNA pieces. These differences are known as restriction fragment length polymorphisms. This technique is also often used to determine an individual’s genotype for a given gene – for instance, to test for the presence or absence of a mutation that confers a certain genetic disorder.

These DNA pieces may then be separated using gel electrophoresis. This method places the DNA sample in a well on one end of a sheet of agarose gel, similar to a thin layer of jello. An electric field is then applied to the gel. Since DNA is negatively charged, it is attracted to the positive end of the field and begins moving through the gel. The larger, longer fragments cannot travel as far through the gel matrix and get trapped near where the sample is loaded. The smaller, shorter fragments can wiggle their way through the matrix more easily and thus travel further. This results in a unique pattern of bands for each individual, depending on the DNA sequence.

Another common method of DNA fingerprinting exploits highly variable repeating sequences called short tandem repeats (STRs). Different people have different numbers of repeat units. For instance, the CSF gene contains the sequence AGAT repeated anywhere between 6 to 16 times. Two unrelated humans will be likely to have different numbers of this AGAT sequence.

While the variable number of repeats displayed at any single STR region will be shared by around 5 – 20% of individuals, if you look at several STR regions simultaneously, this method becomes incredibly powerful. The more STR regions that are tested in an individual the more discriminating the test becomes. In the U.S.A., there are 13 loci (DNA locations) that are currently used for discrimination. This has resulted in the ability to generate match probabilities of 1 in a quintillion (1 with 18 zeros after it) or more. Therefore, it is possible to establish a match that is extremely unlikely to have arisen by coincidence, except in the case of identical twins, who will have identical genetic profiles.

The process begins by extracting DNA from the cells in a sample of blood, saliva, semen, or other appropriate fluid or tissue. Next, each STR region is amplified using polymerase chain reaction (PCR) so that the initially tiny DNA sample is copied many times, creating a sufficiently large pool of DNA for analysis. Finally, the DNA fragments are then separated and detected using gel electrophoresis. Again, the shorter the sequence of repeats, the further the DNA fragment will travel through the gel.

Student Prerequisites
A solid understanding of DNA structure and base pairing rules.