Forensic Technology

Polymorphisms

The difference in nucleotide sequences between humans lies between 0.1-0.4%. That means that people are greater than 99% similar. But when you look around the room at your classmates, you can see that that small difference amounts to quite a bit of variation within our species. The bulk of these differences aren’t even within the coding sequences of genes, but lie outside in regulatory regions that change the expression of those genes. Imagine if there were mutations to the coding sequences, this could be very deleterious to the well-being of the organism. We say that the coding sequences of genes that ultimately lead to proteins has a selective pressure to remain the same. The areas outside of the coding sequences have a reduced and sometimes non-existent selection pressure. These areas are allowed to mutate in sequence and even expand or contract. Areas of changes or differences are called polymorphic (many forms). If you were to read a repetitive set of sequences and count the repetition, you’d make mistakes and lose count. Likewise, DNA polymerase will make errors or stutter in areas of repetitiveness and produce polymorphic regions.

Restriction Fragment Length Polymorphisms (RFLPs)

Restriction fragment length polymorphism (RFLP) is a technique that exploits variations in DNA sequences. DNA from differing sources will have variations or polymorphisms throughout the sequence. Using Restriction Enzymes, these differences in sequences may be teased out. However, if one were to take the entirety of the human genome and chop it up with a restriction enzyme, many indecipherable fragments would be made. In fact, the resulting agarose gel would simply show a large smear of DNA. RFLP analysis requires that a probe to a specific area of DNA be used to identify specific locations. Agarose gels would be transferred to a membrane or filter where they would be hybridized to these radioactive probes.

RFLP analysis was designed for forensic science to discriminate between people. Since people are 2N, they have pairs of homologous chromosomes with the same loci. However, these loci may contain different alleles. In this case, the phenotype for these alleles is the actual sequence that may or may not contain restriction sites. The presence or absence of a restriction site may arise from single nucleotide polymorphisms (SNPs) that reveal the natural variation between people.
The schematic below illustrates a comparison of restriction profiles between two sources. Note that the probe overlaps a restriction site in one of the alleles. This probe will be able to bind to both fragments given sufficient sequence overlap. Upon resolving on an agarose gel, genomic DNA that does not hybridize with the probe will obscure the locus of interest as a large smear. A filter is placed on top of the agarose and pressed against it to transfer the DNA in a process called Southern Blotting. Following a lengthy transfer,  the filter is denatured to and incubated with the radioactive probe. To visualize this probe hybridization, film is exposed to the filter and processed.

RFLPs represent inheritable markers and can reveal relationships between different individuals. A pedigree can illustrate the relationship of the inherited alleles. The technique can be more informative if using multiple probes simultaneously for different loci or to use multi-locus probes that hybridize to multiple locations.

While RFLPs can arise from SNPs, they may also be caused by the expansion or contraction of repeated elements between restriction sites. These repeated elements of DNA are referred to as Variable Number Tandem Repeats (VNTR) and illustrate polymorphisms that normally occur in non-coding regions of the genome.

External Resources

• Using Tandem Repeats for DNA Fingerprinting (interview)
• The first DNA fingerprint (flash simulation)