Sherrie Post¹, Steven Lee¹, Tamyra Moretti², James Robertson², and Bruce Budowle^2
1 California Department of Justice, DNA Laboratory, Berkeley, California
² Forensic Science Research and Training Center, FBI Academy, Quantico, VA

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ABSTRACT

The GenePrint™ PowerPlex™ System (Promega Corporation) is a short tandem repeat multiplex system consisting of two quadruplexes (8 STR loci) that are amplified in one tube. The primers for the loci CSF1PO, TPOX, TH01, and vWA 9CTTv are labeled with the fluorescent dye tetramethylrhodamine, and the primers for the loci D16S539, D7S820, D13S317, and D53818 (GammaSTR™) are labeled with fluorescein. Detection of the products can be achieved either by scanning a slab gel post-electrophoresis or by real-time scanning during electrophoresis as performed by the DNA sequencer.

Due to differences between instruments in the ability to detect the two dyes, PowerPlex™ kits with different concentrations of labeled primers have been developed to optimize signal detection on the DNA Sequencers 373-377 (Applied Biosystems Division, Perkin-Elmer) and FMBIO^®-100 scanner (Hitachi Software Engineering). The performance of a PowerPlex™ beta test site kit was examined on each of the instruments listed. A series of experiments were carried out that involved variation of the template concentration, temperature of annealing, and Mg2+ concentration. In general, the loci appear to be balanced and there were minimal shadow bands (stutter). The performance of TH01 in the PowerPlex™ system was improved over that of the same locus in the original GenePrint™ Fluorescent STR System, because it exhibited a much higher signal intensity in the new system, and stutter at vWA was dramatically reduced.

Using the 373 and 377 DNA Sequencers and 6% PAGE to analyze the PCR products, the signals for the loci were fairly similar in intensity across the loci, except that the signals of some of the loci were more intense than those for D16S539. A major problem with the test kit configuration was that the intensity of the CTTv products was significantly lower than the intensity of the GammaSTR™ products. A higher number of thermal cycles is required to obtain an acceptable CTTv signal relative to the number required for the GammaSTR™ quadruplex and this can lead to saturation of the fluorescein signal and artifactual bands.

Using the FMBIO^®-100 and 6% PAGE for detection of the PCR products the intensity of the CTTv products was significantly lower than the intensity of the GammaSTR™ products, and the signal of D13S317 was slightly lower than the other loci within its quadruplex.

Short tandem repeats (STRs) are made up of repeating sequence units that range in size from 3-7 bps (Edwards et al. 1991, 1992; Hammond 1994). They are highly discriminating, and their small sizes (120-330 bp) allow amplification of several loci simultaneously from degraded samples. For these reasons STRs have become the focus of many studies, and are becoming the favored genetic marker system for forensic casework and databasing.

The Federal Bureau of Investigation (FBI) is currently sponsoring a nationwide STR Standardization Project to identify and validate 12 - 15 STR loci for use in forensic casework and CODIS databasing. The first phase of this project, which has been completed, involved evaluating and optimizing PCR amplification conditions for a number of STR systems. Specifically we examined the signal strength, the balance within and between loci, and the presence of "shadow bands" or stutter, thought to be due to repeat slippage or strand displacement during replication (Weber and May 1989; Schlotterer and Tautz 1992; Sprecher et. al. 1996) for each STR system evaluated.

We carried out a subset of the optimization experiments on the GenePrint™ PowerPlex™ System (Promega) with a beta test site kit and evaluated its performance using fluorescent scanner detection (Hitachi FMBIO^®-100) and automated real time analysis (Applied Biosystems 373/377 DNA Sequencers). The series of experiments which were carried out involved variation of the template concentration, temperature of annealing, Mg2+ concentration, as well as a comparison of DNA polymerase performance.

DNA was extracted from bloodstain samples using a standard organic (phenol/chloroform) extraction procedure and quantitated using the QuantiBlot™ quantitation system. Samples of approximately 2ng template DNA were amplified using either the Hitachi FMBIO^®-compatible, or the Applied Biosystems 373/377-compatible PowerPlex™ beta test site kits. Simultaneous amplification of eight loci (two quadruplexes) was performed using conditions recommended by Promega. Primers for the loci CSF1PO, TPOX, TH01, and vWA (CTTv; Schumm et al. 1994; Ricciardone 1995; Micka et. al. 1996) are labeled with the fluorescent dye tetramethylrhodamine, and primers for the loci D16S539, D7S820, D13S317, and D5S818 (Gamma STR^™; Jim et al. 1996) are labeled with fluorescein. Detection and analysis was achieved using the Hitachi FMBIO^®-100, and the Applied Biosystems DNA Sequencers (373/377) and 6% PAGE.

In the optimization series of experiments, temperatures of annealing ranged from 55 - 64°C, input DNA concentration ranged from 500pg - 5ng, and g2+ concentration ranged from 1.5mM - 3.0mM. In comparing DNA polymerase compatibility with the PowerPlex™ System duplicate samples were amplified with 2 units of AmpliTaq or AmpliTaq Gold™ (Applied Biosystems - Perkin Elmer) per 25µL reaction mix.

Detection of all eight loci of the PowerPlex™ was achieved on both the ABI DNA Sequencers (373-377) and the Hitachi FMBIO^® (Figure 1). The signals for the loci were fairly similar in intensity within each quadruplex (CTTv and GammaSTR™), except that the D16S539 locus had a signal strength approximately 40% lower than the other loci with the ABI test kit, and the D13S317 locus was approximately 10% lower than the other loci of the GammaSTR™ quadruplex when using the Hitachi test kit (Figure 2).

A major problem with both test kit configurations was the difference in intensities between the two quadruplexes. Using the ABI test kit, the intensity of the CTTv products was approximately 50% lower than the intensity of the GammaSTR™ products. A higher number of thermal cycles is required to obtain an acceptable CTTv signal relative to the number required for the GammaSTR™ quadruplex, which could lead to saturation of the fluorescein signal. Using the Hitachi test kit, the signal intensity of the CTTv products was consistently higher than the intensity of the GammaSTR™ products by approximately 50%. The concentrations of the fluorescently labeled primers require further balancing in both PowerPlex™ configuration kits in order to achieve equivalent signal strengths in both quadruplexes. This will allow optimization of the entire octaplex rather than the two quadruplexes independently.

The performance of TH01 and vWA in the PowerPlex™ System was much improved over that of the same loci in the original CTTv GenePrint™ Fluorescent STR System (Promega) because the TH01 locus exhibited a much higher signal strength at the recommended temperature of annealing of 60°C (Promega TMD 006) and stutter at vWA was dramatically reduced (Figure 3). Based on temperature of annealing experiments conducted on the original GenePrint™ CTTv Fluorescent STR System, 62°C was more optimal with respect to signal strength and balance for this system.

PowerPlex™ Optimization Experiments

Temperature of annealing experiments demonstrated that the CTTv portion of the PowerPlex™ could tolerate a wide range of temperatures (55 - 64°C), however, the D7S820 and D13S317 loci of the GammaSTR™ portion decreased in signal strength beyond 60°C, the recommended temperature of annealing (Figure 4).

One to 2ng proved to be the most optimal concentration of starting template with respect to balance, signal strength and minimal stutter for the PowerPlex™ System. Although 5ng of template DNA gave higher signals, there was also increased stutter at the vWA locus. Typing of all 8 loci was achieved down to 500pg, however, differences in signal strength between the quadruplexes could lead to one quadruplex dropping out at a higher level of starting template than the other.

Magnesium is included in the PowerPlex™ buffer system at a concentration of 1.5mM. This concentration appeared to be the most optimal, as added Mg2+ tended to decrease signal intensities (Figure 5).

As recommended by Promega, AmpliTaq™ (Applied Biosystems - Perkin Elmer) rather than AmpliTaq Gold™ (Applied Biosystems - Perkin Elmer) DNA polymerase was used for the amplification of the PowerPlex™ loci. Currently, the buffer system provided with the PowerPlex™ System kits is incompatible with the AmpliTaq Gold™ enzyme, as proved by duplicate samples amplified with the two different enzymes. Promega has anticipated releasing a buffer system in the future, which, with a change in pH, will be compatible with the AmpliTaq Gold™ enzyme.

Initial results indicate that the GenePrint™ PowerPlex™ STR System is an improvement over the original CTTv GenePrint™ Fluorescent STR system with respect to signal strength, balance and stutter. There was very low stutter present in the PowerPlex™ System overall, but there were some signal strength differences between quadruplexes with both kit configurations. In general, the PowerPlex™ System seems to be a useful set for obtaining a lot of data from one PCR, and the products can be analyzed on a variety of fluorescence detection systems.

The authors would like to thank Jay Miller from the FBI for providing support for Sherrie Post during this project, and Martin Buoncristiani, and John Tonkyn from the California Department of Justice for their valuable assistance in support of this research.

REFERENCES

Figure 1. Detection of PowerPlex™ on the Hitachi FMBIO^®-100. Dual color detection of CSF1PO, TPOX, TH01, vWA (CTTv) in red, and D16S539, D7S820, D13S317, and D5S818 (GammaSTR™ in green.) 2ng of DNA amplified at the recommended annealing temperature of 60° C (lanes 9 - 12). 2ng K562 DNA amplified at 60° C (lane 13) and 62° C (lane 14). Scans were performed using 80% laser power and with two different filters: one at 505nm and one at 625nm. The images were then overlaid using dual-color analysis on the Hitachi FMBIO^®.

Figure 2. Loci balance within the GammaSTR™ portion of the PowerPlex™. GammaSTR™ profile obtained using (Top) Hitachi-compatible PowerPlex™ kit configuration, and (Bottom) ABI-compatible PowerPlex™ kit configuration.

Figure 3. Locus dropout and stutter with the CTTv multiplex. 5ng of DNA amplified with the original GenePrint™ Fluorescent STR System CTTv multiplex (CSF1PO, TPOX, TH01, vWA) and detected on the Hitachi FMBIO^®-100. The boxed areas on the gel demonstrate low signal intensities at the TH01 locus, and shadow bands (stutter) at the vWA locus with the recommended annealing temperature of 60° C.

Figure 4. Effect of temperature of annealing on signal intensities of the PowerPlex™. Peak height of the GammaSTR™ portion of the PowerPlex™ plotted against varying temperature of annealing. 2ng of DNA amplified with the Hitachi kit configuration, detected and analyzed on the Hitachi FMBIO^®-100.

Figure 5. Effect of Mg2+ concentration increase on signal intensities of the PowerPlex™. Peak height of the CTTv portion of the PowerPlex™ plotted against MgCI2 concentration. 2ng of DNA amplified with the Hitachi kit configuration, detected and analyzed on the Hitachi FMBIO^®-100.