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By: Curtis Knox and Benjamin Krenke  
Issue: April/May 2007

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. However, many variables can affect the quality and success of an STR analysis.

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 Buel^2,3 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.

Promega has developed a new technology for real-time quantitative PCR. This technology offers an advantage over currently available systems by simultaneously quantitating both total human DNA and male-specific DNA within a sample, in addition to an internal PCR control. This technology, known as the Plexor™HY Quantitation System, will be available in early 2007.

The technology takes advantage of the specific interaction between two modified nucleotides to achieve quantitative analysis.9,10,11As shown in Figure 1, two novel bases, isoguanine (iso-dG) and 5' methylisocytosine (iso-dC), form a unique base pair in double-stranded DNA (Figure 2). To perform fluorescent quantitative PCR using this new technology, one primer is synthesized with an iso-dC residue as the 5'-terminal nucleotide and a 5' fluorescent label; the second primer is unlabeled. During PCR, the labeled primer anneals and is extended, becoming part of the template used during the next round of amplification. During subsequent rounds of amplification, the complementary iso-dGTP, which is available in the nucleotide mix as dabcyl-iso-dGTP, pairs specifically with iso-dC. With the dabcyl and the fluorescent label in close proximity, signal is effectively quenched. This process is illustrated in Figure 2.

Figure 1. Base pairing between isoguanine (iso-dG) and 5´-methylisocytosine (iso-dC).

Figure 2. Quenching of the fluorescent signal
by dabcyl during product accumulation.

Like all qPCR systems, this new technology directly measures the change in fluorescent signal in relative fluorescent units (RFU) at every cycle of the PCR. While existing real-time PCR strategies result in an increased signal as product accumulates, this method results in a decreased signal as product accumulates. Consequently, amplification data continue to present a characteristic three-phase curve (Figure 3).

The part of the curve with the biggest signal change is the exponential phase. The exponential phase is the most consistent phase and is used to estimate the quantity of starting material. An amplification threshold is set within the exponential phase. The point at which an amplification curve crosses that threshold is the cycle threshold (Ct) of the sample. Ct values for a dilution series of a sample of known DNA quantity are used to generate a standard curve, which is used to quantify samples with unknown amounts of DNA (Figure 4).

Figure 3. Plexor HY Autosomal PCR Curves. In the left top win-
dow, amplification curves show the relative fluorescence units
(RFU) at each cycle of the reaction. The amplification threshold
is indicated by a horizontal line across the graph. This thresh-
old is used to establish the cycle threshold (Ct), the cycle at
which an amplification curve crosses the amplification thresh-
old, for each sample. In the bottom left window, melt curves
for each sample demonstrate the dissociation temperature, or
Tm, for the amplified product. A reproducible Tm for each
sample confirms the amplified product is from the intended tar-
get. Similar data are presented for the Y-chromosomal and
Internal PCR Control (IPC) PCR curves (data not shown).

Figure 4. Plexor HY Y
Chromosomal Standard Curve. Using a titration of a human male DNA standard,
amplification curve data (Ct) can be plotted relative to known DNA concentration (ng/µL)
to create a standard curve. The standard curve is used to quantify unknown samples.
The red circles represent standard samples and the blue squares represent unknown samples.
A similar presentation is available for the autosomal analysis (data not shown).

While all real-time qPCR methodologies generate Ct values to quantitate DNA, this method offers the ability to perform a thermal melt curve analysis, which can discriminate between the correct amplification product and nonspecific amplification products and other aberrations. With this technology, fluorescence is quenched when the product is double-stranded due to the close proximity of dabcyl and fluorescent label. Quenching of the fluorescent label by dabcyl is a reversible process, and denaturing the product separates the label and quencher, resulting in an increase in fluorescent signal. Consequently, thermal melt curves can be generated by allowing all product to form double-stranded DNA at a lower temperature (60°C) and slowly increasing the temperature to 95°C. The melt temperature, the temperature at which amplicon disassociation occurs, is dictated by product length and sequence. Thus, nonspecific amplification products will have distinctly different melt temperatures. With some other technologies such as 5' endonuclease assays, the amplification products are destroyed during detection, making melt analysis of the product impossible.

The assay incorporates a number of features from both home-brew and commercially available systems. Both human and male DNA are quantified simultaneously in one assay in addition to an IPC. Human-specific, multi-copy targets are employed to consistently achieve sensitivity of less than 3pg/µl. These features decrease the amount of time and money spent amplifying truly negative samples and allow the user to quickly decide whether to pursue autosomal or Y-STR testing as the most probative test. An internal positive control is included to test for false negatives due to PCR inhibitors. However, this system provides the ability to perform a melt curve analysis at the end of the assay to distinguish specific and nonspecific amplification products.

CONCLUSIONS
Accurate total human and human male DNA quantitation is a necessary step in the process of forensic DNA typing. Without it, DNA analysts can waste time performing STR analysis on unsuitable samples or re-analyzing failed samples. Current methods employed in the field improve on older hybridization-based methods but still leave room for additional enhancements. The Plexor™ HY system incorporates many key features, including high sensitivity, internal positive controls, product melt analysis and simultaneous quantitation of total human and total male DNA.

References:

1. Richard ML, Developmental validation of a real-time quantitative PCR assay for automated quantification of human DNA. J. Forensic Sci.48(5):1041-6. (2003)
2. Nicklas JA, Buel E. Development of an Alu-based, real-time PCR method for quantitation of human DNA in forensic samples. J. Forensic Sci.48(5):936-44 (2003)
3. Nicklas JA, Buel E. An Alu-based, MGB Eclipse real-time PCR method for quantitation of human DNA in forensic samples. J. Forensic Sci.50(5):1081-90. (2005)
4. Walker JA, et al. Human DNA quantitation using Alu element-based polymerase chain reaction. Anal. Biochem.1;315(1):122-8. (2003)
5. Walker JA, et al. Multiplex polymerase chain reaction for simultaneous quantitation of human nuclear, mitochondrial, and male Y-chromosome DNA: application in human identification.Anal. Biochem. 337(1):89-97. (2005)
6. Timken MD, et al. A duplex real-time qPCR assay for the quantification of human nuclear and mitochondrial DNA in forensic samples: implications for quantifying DNA in degraded samples. J. Forensic Sci.50(5):1044-60. (2005)
7. Nicklas JA, Buel E. Simultaneous determination of total human and male DNA using a duplex real-time PCR assay. J. Forensic Sci.51(5):1005-15. (2006)
8. Swango KL, et al. Developmental validation of a multiplex qPCR assay for assessing the quantity and quality of nuclear DNA in forensic samples. Forensic Sci. Int. [Epub ahead of print] (2006)
9. Moser, M.J. and Prudent, J.R. Enzymatic repair of an expanded genetic information system. Nucl. Acids Res. 31(17), 5048–53. (2003)
10. Johnson SC, et al A third base pair for the polymerase chain reaction: inserting isoC and isoG. Nucl. Acids Res.32(6), 1937–41. (2004)
11. Sherrill CB, et al , Nucleic acid analysis using an expanded genetic alphabet to quench fluorescence. J. Am. Chem.Soc.126(14), 4550–6. (2004)

Curtis Knox is the Product Manager for the Genetic Identity group at Promega Corporation which manufactures a complete line of solutions for DNA typing from sample preparation to data analysis. Curtis received his B.S. from Iowa State University. He has over 10 years of experience in the crime lab as a DNA analyst. Curtis can be reached at curtis.knox@promega.com.

Benjamin Krenke is a Senior Scientist in the Genetic Analysis Research and Development group at Promega Corporation. He received his Bachelor’s and Master’s degrees in Molecular Biology from the University of Wisconsin system. He came to Promega after graduation in 1999. His primary focus has been on the development of Genetic Identity products including the Plexor™HY System.