Forensic samples that contain too little template DNA or are too degraded require alternate genetic marker analyses or approaches to what is currently used for routine casework. Single nucleotide polymorphisms (SNPs) offer promise to support forensic DNA analyses because of an abundance of potential markers, amenability to automation, and potential reduction in required fragment length to only 60-80 bp. The SNP markers will serve an important role in analyzing challenging forensic samples, such as those that are very degraded, for augmenting the power of kinship analyses and family reconstructions for missing persons and unidentified human remains, as well as for providing investigative lead value in some cases without a suspect (and no genetic profile match in CODIS). The SNPs for forensic analyses can be divided into four categories: identity-testing SNPs; lineage informative SNPs; ancestry informative SNPs; and phenotype informative SNPs. In addition to discussing the applications of these different types of SNPs, this article provides some discussion on privacy issues so that society and policymakers can be more informed.

Introduction

Genetic typing is a powerful means of establishing identity in criminal cases where biological evidence is found at crime scenes—paternity testing, inheritance matters, identification of victims in mass disasters, and identification of missing persons from human remains. Genetic evidence can be derived from any biological material, including blood, semen, bone, hair, teeth, muscle tissue, and saliva, and can be used for characterization of animals, plants, and microorganisms. The repertoire of genetic markers used for characterization of biological materials has evolved substantially within the forensic field with each marker set and concomitant technology platform, augmenting resolution and/or sensitivity of detection. In this paper we briefly describe the current markers used for forensic identification of humans and also what new genetic markers are likely to be developed, namely single nucleotide polymorphisms (SNPs). SNPs offer further opportunities for the forensic scientist to genetically characterize an evidentiary sample or identify an unknown person.

History of Forensic Genetic Typing

Twenty-five years ago polymorphic protein genetic markers were used to potentially differentiate individuals (1,2,3,; (Figure 1)). However, several factors limited the forensic use of protein-based genetic systems. The discrimination power of these systems is low, so individualization is not possible. Also the proteins are not present at sufficient levels for typing in most tissues, and they are relatively unstable in biological samples exposed to environmental insults.

Figure 1.

Typing genetic polymorphisms at the DNA level helps to overcome these limitations to a much greater degree. First, there is a tremendous amount of variation at the DNA level to exploit for identity testing. Second, any biological material that contains nucleated cells potentially can be typed for DNA polymorphisms. Third, DNA tends to be more stable in forensic samples than proteins. Given the current DNA typing techniques and the battery of available genetic markers, typing of human polymorphisms at the DNA level is more sensitive, more specific, and more informative than the classical protein genetic markers. Moreover, DNA technology affords the forensic scientist the greatest potential to exclude individuals who have been falsely associated with a biological sample and to reduce the number of individuals potentially included as contributors of the sample to a few (if not one) individuals.

The first method used routinely for human identity testing (from the mid-1980s) in the United States and many other countries was restriction fragment length polymorphism (RFLP) typing of variable number of tandem repeat (VNTR) loci (4,5,6). Typing VNTR loci by RFLP analysis provided a very high power of discrimination, but required samples containing at least 10–25 ng of DNA that needed to be relatively intact with fragment lengths up to 10,000 bp for successful typing (7). RFLP analysis was replaced more than a decade ago by amplification of DNA loci by the polymerase chain reaction (PCR) (8) with subsequent typing of genetic markers ((Figure 1)). PCR enables the analysis of samples containing picogram levels of DNA, thus increasing the sensitivity of detection one to two orders of magnitude. In fact, minute quantities of DNA extracted from many types of samples are typed routinely and successfully using PCR-based assays in forensic laboratories. Sample materials include blood, semen, saliva, and sweat deposited on various substrates including clothing, cigarettes, postage stamps, envelope flaps, drinking straws and containers, chewing gum, and face masks; vaginal swabs from a rape victim; various tissues from human remains; and possible reference samples from personal items, such as hair brushes, toothbrushes, and razors, which may be useful in the identification of unknown remains.

The first genetic marker systems analyzed using PCR-based systems were based on SNP variation. Sequence polymorphisms at the HLA-DQA1 locus and polymarker loci (LDLR, GYPA, HBGG, D7S8, and Gc) were typed by use of allele-specific oligonucleotide (ASO) hybridization probes in a reverse dot blot format (9,10,11,12), with a different ASO probe for each allele detected at each locus. The battery of ASO probes was bound to a nylon membrane strip and the PCR-amplified alleles were hybridized to the immobilized probes to which they are complementary. Because an identifier molecule (or tag) was attached to the 5′ end of one of the primers, a detectable label was incorporated into the amplified allelic products. These SNP-based systems offered high sensitivity of detection but did not provide the power of discrimination that VNTR/RFLP typing afforded, and were not very useful for deconvoluting the contributions of mixed samples because of their limited polymorphism.