DNA fingerprinting relies on the unique pattern made by a series of DNA fragments after separating them according to length by gel electrophoresis. DNA samples from different suspects, the victim, and samples from the crime scene are first purified. Restriction enzymes cut the DNA samples into fragments of different lengths. Consequently, the variation in the size of the fragments and hence of their positions on an agarose gel is due to differences in where cutting occurs. Thus differences in patterns between individuals are due to differences in the base sequence of their DNA. The nucleotide differences that cause the fragment lengths to vary are called restriction fragment length polymorphisms. There is believed to be approximately one difference in every 1000 nucleotides between nonrelated individuals. The steps involved in DNA fingerprinting are as follows:
1 . The DNA is cut with a restriction enzyme.
2 . The DNA fragments are separated according their length or molecular weight by gel electrophoresis.
3 . The fragments are visualized by Southern blotting. After transfer of the separated fragments from the gel to nylon paper a radioactively labeled DNA probe is added. The probe will bind to those DNA fragments whose DNA sequences are complementary to the probe.
4 . An autoradiograph is made by covering the blot with radiation-sensitive film. This will show the location of those DNA fragments that reacted with the radioactive probe.
There are many different restriction enzymes, most with unique cutting properties. In practice several different enzymes are used with the same DNA samples, giving different sets of fragments for different people. These can be compared with DNA samples taken from a victim or found at a crime scene. Because there is so much genetic diversity, RFLP patterns from different people can vary a lot. Even if mutations have changed a small percentage of the target sequence around the cut site, there will usually still be enough similarity for binding of the probe to occur. The entire process requires several weeks to finish.
The final product of a DNA fingerprint is an autoradiograph that contains at least five essential lanes. The markers are standardized DNA fragments of known size, which have been radioactively labeled. These help determine the size of the various fragments. The “control” is DNA from a source known to react positively and reliably to the DNA probes and shows whether the test has worked as expected. The experimental lanes have samples from the victim, the defendant, and the crime scene. In this example, blood from the defendant’s clothing was compared with his/her own blood and the victim’s blood. The DNA from the clothing actually matches that of the victim.
Two variants of DNA fingerprinting have been used— single-locus probing (SLP) and multiple-locus probing (MLP) . In SLP, a probe is used that is specific for a single site, that is, a single locus, in the genomic DNA. Because humans are diploid, an SLP probe will therefore normally give rise to two bands from each person for each particular locus. This assumes that the chosen locus shows substantial allelic variation. Occasional persons will be homozygous and hence show only a single band. For full identification using SLPs, it is necessary to run several reactions, each using a different SLP probe. SLP analyses use smaller amounts of material than MLP and are easier to interpret and compare. Statistical analysis and population frequencies are possible using SLP data.
Historically, MLP was used before SLP. In MLP, a probe is used that binds to multiple sites in the genome. Consequently, an MLP probe generates multiple bands from each individual. Because it is not known which particular band comes from which particular locus, interpretation is difficult. Furthermore, statistical analysis is impractical and data cannot be stored reliably in computer databases. In practice, fingerprints generated by MLP probes must be directly compared with others run on the same gel. Consequently, MLP methods have largely been displaced by SLP analysis.
USING REPEATED SEQUENCES IN FINGERPRINTING
A variation of DNA fingerprinting is to look at regions of the DNA that contain variable number tandem repeats (VNTRs) . As discussed in Chapter 8 , this means that sequences of DNA are repeated multiple times and that different people have different numbers of repeats. Repeat sequences vary greatly in length; however, for forensic purposes, relatively short repeated sequences are now generally used and are known as short tandem repeats (STRs) —see later discussion. VNTRs usually occur in noncoding regions of DNA. Hence, using VNTRs protects privacy in the sense that an individual’s coding DNA is not revealed during forensic investigations. VNTRs may be visualized by using restriction enzymes to cut out the DNA segment containing the VNTR, followed, as before, by separation of DNA bands by gel electrophoresis and visualization by Southern blotting. Alternatively, the DNA fragments for VNTR analysis may be generated by PCR (see later discussion). Figure shows corresponding DNA fragments from three individuals who differ in the number of repeats in the same VNTR. Consequently the length of the fragment differs from person to person.
There is often an enormous variation between people in the number of repeats at any particular VNTR site. So there is a very low likelihood of two people matching exactly, or, if you prefer, a high probability they will differ. Some VNTRs have 100 to 200 different variants, making them very useful for forensic analysis. Although VNTRs are not genuine genes, their variants are regarded as alleles and so VNTRs are considered to be “multiallelic” systems.
FIGURE VNTR Fingerprinting
Genomic DNA has regions with repeated sequences. In each individual, the number of repeats varies, and therefore the lengths of these regions can be compared to distinguish identities. The repeated region is isolated using restriction enzymes from three individuals marked A, B, and C. The fragments are run on agarose gels to compare the lengths.
One practical problem here is that multiple tandem repeats give so many closely packed bands that standard agarose gel electrophoresis cannot discriminate the different fragments. Different types of gels such as polyacrylamide may resolve closely spaced bands. Alternatively, the fragments can be separated on a gradient gel.
The original DNA fingerprints, invented by Alec Jeffreys in England in 1985, used highly variable VNTRs with long repeat sequences. DNA was isolated and cut with restriction enzymes because PCR had not been invented. The cut fragments were probed using MLP to generate the early DNA fingerprints.
The STR (short tandem repeat) is a subcategory of VNTR in which the repeat is from two to six nucleotides long. Most STRs are not as variable in the number of repeats as VNTRs with longer unit sequences. Many STR sequences have only 10 to 20 alleles and hence cannot provide unique identification alone. However, many STR loci are available, and if several are analyzed simultaneously, this will provide enough data that the pattern would be unique for each individual. Today, STR analysis is done using PCR to generate the DNA fragments.
TRACING GENEALOGIES BY MITOCHONDRIAL DNA AND THE Y CHROMOSOME
Mitochondrial DNA sequences have been very useful in tracing the recent evolution of the human species at the molecular level. Analysis of mitochondrial DNA (mtDNA) can also be used in forensics. The main advantage is that mitochondrial DNA is present in multiple copies per cell and so is relatively easier to obtain in sufficient amounts for analysis. The sequence of mtDNA varies by 1% to 2% between unrelated individuals.
The major disadvantage is that mitochondrial DNA does not vary between closely related individuals. Mitochondria are inherited maternally, and mitochondrial DNA sequences are therefore shared among groups of people derived from the same maternal lineage. If two samples of DNA show different mitochondrial sequences, this indicates that they come from different people. However, the opposite is not true. Identical mitochondrial sequences are found in people related on the mother’s side.
Mitochondrial DNA has been used to derive family ancestries. Indeed, it is now possible to submit personal samples of DNA for analysis to companies such as Oxford Ancestors. Their MatriLine service allows persons of European descent to trace their maternal ancestry back to one of seven ancestral females. Almost everyone in Europe, or whose maternal roots are in Europe, is descended from one of only seven women whose descendants make up well over 95% of modern Europeans. For genealogical purposes, each of these seven women may be regarded as the founder of a “maternal clan.”those whose maternal roots lie outside Europe, a similar analysis is available, but is not yet so detailed.
In contrast to mitochondrial DNA, the Y chromosome follows a paternal pattern of inheritance. The Y chromosome contains many STR sequences in noncoding regions. However, most have few different alleles, and so only a few are suitable for forensic analysis. One advantage of using Y-linked STR loci is that any sequence specific to the Y chromosome must have come from a male. This is often useful in cases of sexual assault.