DNA Profiling: State of The Art

Since its development in the mid-1980s (1), advancements in forensic DNA analysis were characterized by a continuous increase of sensitivity and discrimination power. This process was paralleled by a size reduction of the investigated DNA fragments in order to comply with the requirement to type severely degraded DNA in challenging forensic samples (2). The most powerful method that combines the aforementioned requirements is the analysis of autosomal short tandem repeats (STRs). Forensic relevant STRs are DNA segments that are typically found in noncoding regions of the human genome and are composed of repeating units of tri- to pentanucleotide sequence motifs. The elevated mutation rate of STRs has led to a high degree of polymorphism in humans, which renders STR typing powerful for identity testing (3). Their relative small amplicon size (100–400 bp) and their amenability to multiplex analysis have rendered STRs the most important forensic DNA markers. The establishment of a laboratory-independent nomenclature (4) allowed for the direct comparison of typing results across borders and set the basis for the foundation of national DNA databases that are successfully used as intelligence tools by executive forces. Nowadays, national DNA databases contain millions of STR fingerprints that effectively help to link an unknown stain to the perpetrator. The STR fingerprint that contains the evidential information consists of the combined genotyping information obtained from a selected number of well-characterized and validated STR loci. Harmonization of technology and of STR markers has thoroughly been pursued by international forensic scientific working groups such as the European DNA Profiling Group (EDNAP; www.isfg.org/ednap/ednap.htm) and the European Network of Forensic Science Institutes (ENFSI) DNA Working Group (www.enfsi.org/ewg/dnawg). This has led to the selection of core loci that were adapted by the forensic community and constitute the basic configuration of the national DNA databases. The International Standard Set of Loci (ISSOL) that is recommended by the Interpol DNA Monitoring Expert Group (www.interpol.int/Public/Forensic/DNA/DNAMEG.asp) involves the STR loci vWA, TH01, D21S11, FGA, D8S1179, D18S51, and D3S1358. Depending on the typing chemistry that is used by the laboratory, the following STR loci add to the standard set: D2S1338, D19S433, D16S539, D7S820, D13S317, D5S818, CSF1PO, Penta D, Penta E, TPOX, and SE33 (www.interpol.int/Public/Forensic/dna/HandbookPublic.pdf). In a recent attempt to extend the discrimination power of STR profiles for samples containing heavily degraded DNA, so-called miniSTRs have been evaluated and suggested as additional loci (D2S441, D10S124, D22S1045) (5).

STR typing is usually accomplished via selective amplification using PCR and consecutive electrophoretic sizing of the amplified fragments. Their size is determined via the comparison of observed migration times to those of size standards. The individual alleles are denoted by comparing their migration times to those of the allelic ladder, a selection of sequenced allele variants that need to be co-analyzed with the samples in question. So far, capillary electrophoresis (CE) with multicolor fluorescence detection represents the method of choice for STR typing, as it offers 1-bp resolution for the discrimination of all allelic length variants within an STR fingerprint. Length variants have been rigorously studied in all world populations. This serves as the basis for the calculation of combined allele frequencies that are used to determine statistical values to support the weight of evidence (e.g., www.strbase.org). STR amplicons, however, may contain more discriminative information than just the fragment length. This has been shown for selected STR alleles by direct sequencing analysis (6,7,8,9) and the identification of small haplotype blocks in which SNPs are tightly linked to the STRs (10,11,12). Based on sequencing experiments, STRs were classified as simple (repeats that contain only units of identical length and sequence), compound (repeats that comprise two or more adjacent simple repeats), and complex (repeats that contain several repeat blocks of variable unit lengths along with more or less variable intervening sequences), which indicates that there is additional sequence variability in STRs that would allow for discrimination of fragments with identical length. There is no doubt that sequencing of STRs would not substitute the established fragment-length analysis for multiple reasons, including hands-on time and cost. However, a method that is capable of discriminating sequence differences in STR amplicons would be beneficial for a number of forensic applications.