DNA – an introduction part 2

Having arrived at this point from consideration of the basics of genetics and the biochemistry of DNA, you may think that the DNA typing of blood for forensic identification is a matter of identifying the alleles in selected genes. But it isn’t. One of the more recent advances in genetics was the successful sequencing of the total human genome. We now know that there are only between 20,000 and 25,000 genes in the human genome, considerably fewer than was expected. Genes account for less than 2% of the total, the remainder being non-coding DNA. Current research indicates that most of the non-coding DNA has a biological function, including regulating gene expression.

About 8% of the genome consists of what is termed repetitive DNA sequences, which are head-to-tail or tandem repeats of nucleotide sequences. More than 10,000 have been identified, each being characterised by the identity and number of nucleotides in the base unit of the sequence. Their value as a forensic marker arise because some have alleles that code for variation in the number of repeats of that sequence. It is these tandem repeats that are the basis of most of today’s DNA testing, specifically Short Tandem Repeats, or STRs, also called microsatellites. The STRs used in forensic identification are mostly sequences that are four bases long, called tetranucleotides. The alleles consisting of the sequence repeated 3 to 25 times. Not all are tetranucleotides.

And some of the ones in common use have an allele range of up to 42. The first recorded case where DNA was used is that of Colin Pitchfork in England in 1987. Police investigating the related rape and murder of two 15-year-old girls in Leicestershire in England contacted Dr. Alec Jeffreys at the University of Leicester for assistance. They requested that he applied his newly discovered DNA profiling method to the analysis of the semen on the vaginal swabs from the victims and compare the results with samples given voluntarily by 5,000 men living in the surrounding areas. Eventually, a match was obtained to Pitchfork, who was subsequently convicted of the offences. Dr. Jeffreys did not use STR analysis.

It was not available at that time. The technique that he used, and which launched forensic DNA analysis, differed in two significant respects. The target stretches of DNA that he used, called minisatellites, were much longer than the STR microsatellites. And the technique employed to identify the size variations, called Restriction Fragment Length Polymorphism, or RFLP, was more cumbersome and lengthy. The tool that made forensic STR analysis possible is the Polymerase Chain Reaction, or PCR. Around the same time that Alec Jeffreys was working with minisatellites, a number of other researchers were working with the enzyme DNA polymerase, which will synthesise DNA in cells by copying the base sequence of a template DNA strand. Kary Mullis was one of them.

And in 1983, he realised that a pair of primers could be used to bracket a target DNA sequence which could then be copied using DNA polymerase. He published the work in a joint paper with colleagues in 1986. Mullis was awarded the Nobel Prize in chemistry in 1993 for his work along with Michael Smith. Although as with so many discoveries, including the 1962 Nobel Prize in physiology or medicine awarded to Crick, Watson, and Maurice Wilkins, there were many others whose earlier or contemporaneous contributions are noteworthy. The amount of DNA in even a very large bloodstain is far too small to permit direct analysis of the STRs in it. And that’s where PCR comes in.

The process targets a specific STR in the DNA molecule and then amplifies it by more than a million times. In this way, the tiniest of blood or semen stains can be typed. Most forensic laboratories use between 10 and 20 different STRs to build a DNA profile. The names, structure of the core sequence, and the allele ranges for some STRs can be seen in the table. You will see that there are two different types of name. THO1 is associated with the tyrosine hydroxylase located on chromosome 11. And TPOX is associated with the thyroid peroxidase gene on chromosome 2.

The others in the table follow a standard naming convention, where D1 signifies a location on chromosome 1, D13 on chromosome 13, and D16 on chromosome 16. And the S designation refers to the sequence on the chromosome where the allele is located. Each of the STRs used in forensic profiling is located on a different chromosome from any of the others. As we shall see, this allows the frequency in the population of the various allele combinations to be calculated.