Multiplex STR analysis has long been accepted as the gold standard in the field of human identification. This method is highly informative, allowing DNA identification to be made with a high degree of accuracy.
To provide optimal results, STR assays require a defined range of template material. The most commonly available STR megaplexes for forensic analysis have been optimized for template input of 0.5-1.5ng of DNA. Using template quantities less than the suggested range increases the likelihood of allelic imbalance, and partial amplification. Conversely, too much template can lead to signal saturation, nonspecific amplification, and imbalance between loci. Accurate quantitation of template DNA is necessary for optimal STR results, while conserving limited DNA material. This reduces the need for re-amplification and corresponding costs associated with additional analyses.
Several methods can be used to quantitate DNA prior to STR analysis. Older methods currently in use have various shortcomings. For example, hybridization-based methods can produce increased levels of false-negative results, due to the lack of sensitivity. Additionally, hybridization results can be subjective, as interpretation is done by visual comparison of band intensities.
Amplification-based quantitation methods offer improved sensitivity over hybridization-based methods, and real-time PCR methods offer the greatest dynamic range. Finally, the numerical output of real-time or quantitative PCR (qPCR) increases the objectivity of data interpretation.
CURRENT STATE OF qPCR IN FORENSICS
DNA-typing laboratories that wish to take advantage of qPCR have only a few options. They may purchase a commercially available kit, or they may construct their own “home-brew” system using various methods.
Forensic laboratories have a rich history of creating home-brew systems that fulfill the specific needs of the testing facility. These home-brew systems use commercially available qPCR chemistries available to research labs. These methods include 5' endonuclease assays and double-stranded DNA-binding dye-mediated qPCR. Laboratories generally design primers or probes for human DNA targets of their choosing and optimize the system until a valid assay is realized.
Home-brew methods have been developed for both single and multiplex human targets. The method described by Richard et al. targets the flanking region of one of the standard STR markers currently used in forensic testing, the TH01 locus.1 Other single-target assays for the highly repetitive Alu family of repeats have been described by Nicklas and Buel2,3 and Walker et al.4 The use of multi-copy targets such as Alu sequences enhances sensitivity of the system and thus has an advantage over single-copy STR targets. Maximizing sensitivity of the quantitation is a major issue for STR-typing laboratories, as popular hybridization-based methods can miss low levels of DNA that are adequate to generate usable STR profiles. More recently, multiplex approaches to forensic DNA quantitation have been reported. Simultaneous quantitation of human nuclear DNA (nDNA) and mitochondrial DNA (mtDNA),5,6 nDNA and human male-specific Y-chromosome DNA7 and triplex analysis of nDNA, mtDNA and human male DNA5 have all been demonstrated. Swango et al. describe the use of qPCR not only to quantitate DNA, but also to determine DNA quality by comparing similar nDNA targets with different ampli-con lengths.8 The benefit of multiplexing is clear in the variety of questions that can be answered from the same analysis.
The disadvantages of home-brew systems are readily seen. The manufacturing of reagents within a practitioner laboratory is time-consuming and requires extra quality-control steps. This time could be used more efficiently testing samples. Also, many home-brew systems lack some desirable features such as A) internal positive controls (IPC) for PCR inhibitors, B) the necessary sensitivity or C) a suitable configuration to answer the most necessary quantitation questions in one multiplex.
The currently available commercial total human and male DNA assays contain an internal positive control to help test for PCR inhibition. These assays amplify single-copy targets for both human autosomal DNA and Y-chromosomal DNA. Similarly, the STR assays that rely on these quantita-tion results amplify single-copy targets throughout the human genome. However, quantitation systems are designed to use a minimal volume of DNA template, often 2µl, to conserve material, whereas STR assays such as the PowerPlex®16 System can accommodate a much larger volume of DNA template. Thus, sensitivity is an important consideration when choosing a quantitation assay due to the limited input volume. For example, given the sensitivity of current STR assays, with users performing low-copy-number (LCN) sample analysis with less than 50pg of total input DNA (2.5-5pg/µl), the quantitation assay should achieve reliable detection of as little as 5-6pg/2µL to provide confidence when deciding whether template levels are sufficient. The sensitivity of current commercial kits with single-copy targets may be inadequate for confident determination of no DNA in LCN sample situations. Use of a system with greater sensitivity would provide greater confidence in the decision to proceed or not with potential LCN samples. The most important questions for almost all evi-dentiary samples are: 1) how much total human DNA is in a sample, 2) how much male DNA is in a sample and 3) is PCR inhibition affecting these quantitation results. A multiplex assay should address these questions simultaneously and reduce the labor involved in multiple analyses of the same sample.