separating the X from the Y
31 May 2006 by Evoluted New Media
Finding the needle in the haystack: Male-specific DNA in forensics
Finding the needle in the haystack: Male-specific DNA in forensics
In criminal investigations, sexual assault crimes represent a significant portion of the casework involving biological evidence analysed in forensic laboratories. A ten-year study shows that, while the number of reported sexual attacks in England and Wales is increasing, the conviction rate remains low and the number of convictions over the study period has stayed relatively constant1. The rapidly increasing use of DNA analysis since the mid 1980s has made genetic profiles decisive evidence during trials for crimes of all types.
Modern forensic techniques for DNA profiling are based on the analysis of short tandem repeats (STRs), repeated sequences of four to five bases of DNA and, since the mid 1990s, STR analysis has become widely used for DNA genotyping for forensic and other applications for its sensitivity and discriminatory capacity. However, there are several complex and specific challenges in the identification, isolation and analysis of male DNA in biological material containing mixtures of male and female DNA. Traditional methods of segregating male DNA in biological evidence from sexual crimes have involved separating the spermatozoa from other cells, thereby fractionating male and female DNA for subsequent analysis using standard STR typing methods. This differential extraction is both time-consuming and inefficient and, due to the typically high quantity of female DNA in sexual assault evidence, the success rate of detecting the minor male component fails in 20 - 30% of cases. Furthermore, this approach is unable to obtain a male profile of vasectomised perpetrators or from azoospermic semen, where no sperm cells are present for separation and analysis. It is also unable to identify low levels of male DNA in other material encountered in evidence obtained for violent crime, such as skin cells found on weapon handles and in fingernail scrapings. Problems also arise in cases where there are multiple perpetrators, resulting in complex mixtures.
The difficulty of analysing male DNA in mixed samples is illustrated by the example of the conviction in the US of Timothy Durham in 19932. Durham was convicted by jury and sentenced for 3,000 years (!) for the rape of an 11 year old girl in Oklahoma, despite 11 witnesses supporting his alibi placing him in another state at the time of the attack. Evidence against him reportedly matched Durham’s DNA to that of the semen donor. Post-conviction testing, however, indicated that the initial test was a false positive. It was revealed that the original DNA test was sufficiently contaminated with the victim’s DNA, so that the victim’s alleles combined with those of the true rapist produced an apparent match to Durham’s genotype. Numerous subsequent tests confirmed his continued claims of innocence, and eventually led to his release in 1997.
The fact that only 5% of the Y chromosome is homologous with the X chromosome, leaving a large portion of the Y chromosome that is male-specific, offers a potential for the development of methods to profile male DNA within mixed-sex DNA samples. A few STRs had already been identified on the Y chromosome (approximately 20 in 2000), including one which was used in a forensic investigation in 1992, which ultimately excluded a jailed subject from a case of murder3. However, it was not until recently, with the sequencing of the euchromatic region of the Y-chromosome in 20034, that a substantial number (over 200) of male-specific STR loci were identified. Of these, a selection of core Y-STR loci have been designated as universally accepted genotyping loci. The polymorphisms at these loci have been well characterised and are already in use in forensic laboratories worldwide, not only for genetic profiling of male perpetrators in crimes, but also in cases of disputed paternity.
During the last five years, several genotyping systems have been designed to analyse and profile Y chromosome DNA, based on the amplification and genotyping of Y-STRs. The AmpFlSTR Yfiler PCR Amplification Kit, developed by Applied Biosystems, was released in 2004 and is the latest in a line of this type of genotyping systems, which profiles the STRs on the Y-chromosome by a multiplex PCR assay, co-amplifying 17 Y-STRs in a single reaction. The 17 loci include the core set defined as the European Minimal Haplotype, another subset of loci recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM), plus six additional highly polymorphic loci to increase the discrimination capacity of haplotype analysis. These kits provide a solution to the problem of detecting low levels of male DNA within a high female DNA background; the Yfiler system, for example, can routinely detect male DNA in samples containing greater than 1:1000 male:female mixture ratios5. This is, of course, particularly important for sexual assault cases where conventional autosomal DNA genotyping methods would not be able to resolve male DNA. The PCR also makes the kit extremely sensitive, allowing complete male DNA profiles from as little as 125 picograms of input DNA5. Multiplex Y-STR also has sufficient discriminatory capacity to help in sexual assault cases involving multiple male perpetrators.
Y-STRs not only offer the potential to improve the chances of obtaining DNA profiles of male perpetrators of sexual assault, it could also be useful to analyse evidence in cold cases, where conventional DNA methods of the time had failed. In a case in 19986, Y-STRs were profiled from 25-year-old evidence collected from two rape cases in Japan, in response to a retrial request by a condemned criminal. The Y-STR alleles from the vaginal swabs analysed were found to be identical to those of the accused criminal, confirming the original verdict, and his retrial request was rejected.
Together with the increased use of Y-STRs in forensic applications as well as in studies of population genetics, online databases have been created to allow the sharing of Y-STR profiles, called haplotypes, obtained from numerous studies. The Y Chromosome Haplotype Reference Database (YHRD, www.yhrd.org), now containing nearly 40,000 haplotypes, was established in 2000 specifically to generate reliable Y-STR haplotype frequency estimates to be used in the quantitative assessment of matches in forensic and genealogical casework. This type of database permits forensic scientists to estimate the frequency of a particular Y-STR haplotype, which is an important consideration in forensic applications as, unlike autosomal STRs, the unique biology of the Y chromosome prohibits the extrapolation of the discriminatory capacity by the product rule, simply multiplying the probabilities of the match at each locus. The Y chromosome is inherited from father to son so, barring mutations, there is no recombination of a set of alleles, and male relatives share the same Y-STR profile. Thus, a commonly accepted means of estimating the frequency of a Y-STR haplotype is via the counting method of available information in databases. This also means that other highly polymorphic Y-STR loci, additional to the established core loci, (such as the six extra loci in the Yfiler system) may be invaluable in further increasing the discriminatory capacity of a multiplex Y-STR analysis.
Conclusions
Modern forensic techniques are allowing increasing use of evidence based on DNA profiles for legal cases, and are now playing a significant part in the conviction or acquittal of defendants. However, complexities remain in identifying, isolating and analysing male specific DNA in mixed samples containing female DNA. The identification of Y-STRs and recently developed genotyping systems are now addressing these problems, with sensitive kits available that allow male DNA profiles to be obtained from minute and highly contaminated samples. The analysis of Y-STRs is undoubtedly proving to be a valuable DNA typing tool in the arsenal of modern forensic scientists.
By Sue Ann Molero, Applied Biosystems
References
1 Cross-National Studies in Crime and Justice (2004) United States Department of Justice: Bureau of Justice Statistics
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5 Mulero JJ, Chang CW, Calandro LM, Green RL, Li Y, Johnson CL, Hennessy LK (2006) Development and validation of the AmpFlSTR® Yfiler™ PCR Amplification Kit: a male specific, single amplification 17 Y-STR multiplex system. Journal of Forensic Science 51(1): 64-75.
6 Honda K, Roewer L, de Knijff P (1999) Male DNA typing from 25-year-old vaginal swabs using Y chromosomal STR polymorphisms in a retrial request case. Journal of Forensic Science 44(4): 868-872.
