James W. Schumm, Ann M. Lins, Katherine A. Micka, Cynthia J. Sprecher, Dawn R. Rabbach,
and Jeffrey W. Bacher.
Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711 USA.

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ABSTRACT

The era of developing criminal population databases to link suspects to crime scenes and crime scenes to one another by evidence comparison is upon us. To achieve the ultimate benefit of this approach, hundreds of thousands to millions of individuals, primarily convicted criminals, will be typed using polymorphic short tandem repeat (STR) systems. The enormous labor involved with this effort can be minimized by using multiplex STR systems which are detected using semi-automated data collection instruments.

• We have developed three fluorescein-labeled STR quadriplex systems, each with the following properties.
• Within each quadriplex, no alleles of one STR locus overlap in size with alleles of another locus.
• Fluorescent allelic ladders for each locus have been developed.
• All loci except vWA are simple STR systems. Microvariants have been observed with only two of the twelve loci (i.e. TH01 allele 9.2, TH01 allele 8.3, F13A01 allele 3.2).
• Each system is compatible for use with the following instruments:
• Hitachi Software Engineering FMBIO^® and FMBIO^® II Fluorescent Scanners (San Bruno, CA)
• Molecular Dynamics FluorImager™ SI and 595 Fluorescent Scanners (Sunnyvale, CA)
• Applied Biosystems 310 Capillary Electrophoresis Unit (Foster City, CA)
• Applied Biosystems 373 and Prism™ 377 DNA Sequencers (Foster City, CA)
• Genomyx LR™ DNA Sequencer (Foster City, CA)
• Pharmacia A.L.F. DNA Sequencer (Uppsala, Sweden)

By labeling one of the quadriplex primer sets, CTTv, with a rhodamine derivative, and adjusting other parameters, we have created the GenePrint™ PowerPlex™ System which allows simultaneous amplification and detection of eight STR loci. This system has enormous discrimination power arising from the combination of seven simple STR loci plus the compound locus, vWA, described in this work.

Different versions of the PowerPlex™ System can be used effectively with the Applied Biosystems instruments described above or the Hitachi FMBIO^® Fluorescent Scanner. With the ABI Prism™ 377 DNA Sequencer, it is possible to run two gels during the work day and one overnight. With the Hitachi fluorescent scanner, 20 to 30 gels can be run in standard rigs, then analyzed sequentially in the scanner during the day. In the latter example, this combination of reagents and instrumentation provides the necessary ingredients to achieve the throughput which will be required to generate large population databases efficiently.

Governments throughout the world have committed themselves to improve analysis of crime scene evidence and its use in identification of crime scene participants. Part of this effort includes the establishment of national databases which will contain DNA profiles of individuals convicted of selected crimes. Short tandem repeat (STR) polymorphisms (Edwards, et al., 1991, 1992, Sprecher, et al., 1996) are ideal markers for use in databases. When selected properly, they provide rapid analysis of small amounts of sample material with precise and accurate allele assignment. The fluorescent STR systems described in this work have been selected for their superior quality in these attributes.

By developing multiplex STR systems (i.e. combinations of STR loci which are amplified and analyzed simultaneously), we have substantially decreased the work required to analyze samples, while increasing the power of discrimination of the method. In this work, we will describe the GenePrint™ PowerPlex™ STR System which, alone, has discrimination power adequate for national database searching as well as the even more demanding statistical requirements of paternity analyses.

Selection of instruments for STR analysis will also be discussed. In brief, a multi-color fluorescent scanner for detection of STR loci offers high throughput and the greatest convenience to the practitioner.

Use of fluorescent-labeled primers offers several advantages over silver stain analysis (Bassam et al., Promega Technical Manual #TMD004) to detect unlabeled systems. First, with prelabeled primers, the amplification products contain a fluorescent dye for immediate detection. There is no need for separation of the glass plates which contain the gel prior to detection, eliminating one step which occasionally results in destruction of the gel. The staining procedure is also eliminated, saving a significant amount of time and avoiding waste products of the silver stain method.

Furthermore, some artifacts which are observed with the silver stain method are masked in the fluorescent method. For example, with the silver stain method, the opposing strands of an amplification product sometimes appear as a doublet in the denaturing gel systems used for fragment separation. This occurs because while being the same length, the products contain complementary, not identical, sequences. This sequence variation can affect migration rates. With the fluorescent systems, only one primer in each primer pair is tagged with a fluorescent dye. Thus, only one of the strands of the amplification product is visualized by the detection instrument, providing a cleaner image.

The loci described in this work were also chosen for a minimum of Taq DNA Polymerase-related artifacts, (1) repeat slippage (Levinson and Gutman 1987, Schlotterer and Tautz 1992), and (2) terminal nucleotide addition (Clark, 1988, Hu, 1993, Magnuson et al., 1996, Walsh et al., 1996), and a genetic artifact, microvariant alleles. Repeat slippage, sometimes called "n-4" or "shadow banding," occurs as loss of a repeat unit during DNA synthesis through regions of repeated sequence. The amount of this artifact observed is dependent primarily on the locus and the DNA strand (i.e. sequence) being replicated. We have chosen loci with little to no repeat slippage. The vWA locus is an exception, revealing as much as 10% stutter. It was chosen primarily for its popularity in the forensics community.

Terminal nucleotide addition is another property of Taq DNA Polymerase. In this case, the polymerase comes to the end of an amplified fragment, then adds a nucleotide, generally adenine, as an extra base. This activity often does not occur with 100% efficiency and varies with different primer sequences. Thus, an artifact band one base shorted than expected (i.e. missing the terminal addition), is sometimes seen. Redefinition of the primer sequences and/or adding a final extension step in the amplification protocol (60°C for 30 minutes, Walsh et al., 1996) can lead to conditions of essentially full terminal nucleotide addition. We have observed that rules proposed to describe primer sequences for complete addition do not completely agree with our experience (data not shown).

One other artifact seriously considered during our selection process was the presence of microvariants (i.e. alleles differing from one another by lengths shorter than the repeat length). In general, microvariant alleles complicate separation, interpretation, and assignment of alleles. While it is possible to identify highly polymorphic markers with relatively low presence of stutter bands and terminal nucleotide addition artifacts (Sprecher et al., 1996, Lins et al., 1996), there appears to be a correlation between a high degree of polymorphism, a tendency for microvariants, and increased mutation rate (Moller et al., 1994; Brinkmann et al., 1995). Increased mutation rates, of course, complicate paternity analyses, including those in forensic casework. For these reasons, we have limited our present work to loci with moderately high polymorphism and only two of the twelve loci

selected for our work (See Table 1), TH01 and F13A01, contain microvariant alleles. We have developed these specially selected loci into multiplex systems, described below, which maintain the positive qualities of easy, reliable interpretation, while adding very powerful means for discriminating individuals.

Fluorescent detection of STR loci has been assisted by recent advances in instrumentation. While it is possible to conceive of employing ethidium bromide staining and transilluminator illumination for this work, the multi-color detection of pre-labeled amplification products provides for higher throughput and diminution of the visualization of artifacts described in the preceding paragraphs. A substantial initial investment is required to attain this level of sophistication using either fluorescent scanners or DNA sequencers.

Our laboratory has three separate instruments for fluorescent STR analyses – the Molecular Dynamics (MD) FluorImager™ SI fluorescent scanner, the Hitachi FMBIO^® fluorescent scanner, and the Applied Biosystems (ABI) Prism™ 377 DNA sequencer. These instruments differ from one another in several respects. First is the number of distinct fluorescent dyes which can be discriminated with each instrument. The MD instrument can detect several fluorescent dyes, but distinctly separates only one. The Hitachi instrument is capable of separating three colors into distinct data, while the ABI machine can be used to separate four. A new MD instrument, the FluorImager™ 595 fluorescent scanner has recently been released. There are reports that this instrument can cleanly separate two fluorescent dyes used in STR analyses.

From this analysis, you might think that the ABI instrument offers the highest throughput. In fact, precisely the opposite is true. This results from the difference between the DNA sequencer and the fluorescent scanners. With ABI DNA sequencers, it is necessary to dedicate the machine to electrophoresis of each gel. This means that the instrument is unavailable for other uses while each gel is pre-run and run. In practical terms, without resorting to tricks of some sort, we find that we can run two ABI gels during the day and one overnight, with proper coordination of personnel.

By contrast, the fluorescent scanners from both MD and Hitachi allow you to run several gels simultaneously on the bench top (i.e. away from the scanners themselves). As electrophoresis of each gel is completed, the gel is placed into the instrument which then scans it to identify the location of fluoresceinated materials. A digital computer image of the gel is generated for display and line tracings may be prepared for additional analysis. This approach offers the potential to analyze 25 or more gels in a single day.

In addition to high throughput, the fluorescent scanners offer convenience. For example, laboratories often run three gels per day for casework or research. With the gel-dedicated approach of the ABI instrument, this requires a full 24 hours with three separate set up and break down periods. The scanners, on the other hand, allow the same analyses to be performed in a single morning as all three gels may be run simultaneously, then scanned in rapid succession to complete the work. Fluorescent scanners also offer a flexibility not attained with the DNA sequencer, as they may be used to analyze ethidium bromide stained gels or microtiter dishes containing fluorescent materials. The combination of distinct three-color detection and fluorescent scanning makes the Hitachi FMBIO^® instrument especially attractive in our experience.

The major limitation in using STR systems has been that STRs which display few or no microvariants and low mutation rates are not so polymorphic as the best of the VNTR markers. Thus, there is a need to develop high throughput approaches with STRs to overcome this deficiency. Our selection of individual STR loci each with a limited size range of known alleles (Table 1) means that several STR systems may be detected simultaneously in limited and well-defined regions of the same lane on a gel. This offers the potential to develop STR multiplex systems.

We have developed three fluorescent quadriplex STR systems, CTTv, FFFL, and GammaSTR™, to satisfy the need for high throughput analyses (Table 2). As illustrated in Figure 1, each quadriplex allows co-amplification of four STR loci and each system contains a mixture of allelic ladders for the four loci. The allele size ranges of the different loci in an individual multiplex do not overlap one another allowing for rapid and confident typing of alleles (Figure 1).

*FL 5'-terminal fluorescein label

**TMR 5'-terminal carboxy-tetramethylrhodamine label

All three multiplexes use the same amplification protocol, allowing simultaneous amplification in a single thermal cycler. Amplification of less than or equal to 1ng of DNA template is readily detected for each of these systems (data not shown). Because one primer from each locus is labeled with fluorescein, detection of the amplified products may be achieved using any of several fluorescent imaging instruments. While Figure 1 displays amplification products of each multiplex using the Molecular Dynamics FluorImager™ SI fluorescent scanner, the

same materials may be detected using the Hitachi Software Engineering FMBIO^® fluorescent scanner, the Pharmacia A.L.F. DNA Sequencer (The A.L.F. Express requires use of a different dye), or the Applied Biosystems (ABI) 373 or 377 DNA Sequencer. These multiplex systems are not compatible with the Hitachi Electronics Engineering DNA Sequencer which requires a different fluorescent dye for detection. Instrument compatibility of the various one- and two-color multiplexes is summarized in Table 3.

Table 3. Instrument Compatibility with GenePrint™ Fluorescent STR Systems.

User = Recommended by other users. Not guaranteed for optimum performance by Promega.

+ = Qualified for this use in Promega laboratories following manufacture.

For those fluorescence detection instruments which are capable of simultaneously detecting and discriminating more than one fluorescent dye, we have developed a multiplex system which permits co-amplification and two-color detection of eight STR loci. This system, known as the GenePrint™ PowerPlex™ System contains a mixture of eight primer pairs, provided as a single pre-mixed and pre-qualified cocktail. One primer of each pair has been labeled with carboxy-tetramethylrhodamine (TMR) for the loci present in the CTTv multiplex, while one primer for each locus in the GammaSTR™ system is labeled with fluorescein. All eight loci are amplified simultaneously in a single tube and analyzed in a single gel lane as illustrated in Figure 2 for the Hitachi FMBIO^® fluorescent scanner and in Figure 3 for the ABI Prism™ 377 DNA Sequencer. Amplified samples are compared with a mixture of the eight corresponding allelic ladders to determine allele content of unknown samples rapidly and precisely. Different primer cocktails and allelic ladder mixes have been developed to accommodate the Hitachi Software Engineering fluorescent scanner and the ABI DNA sequencers, respectively. This was necessary because the different lasers in the two instruments activate the fluorescence TMR and FL dyes with different relative efficiencies.

Recently, we have developed a new size marker for use with the multiplex system. The 16 fragments of this marker are evenly spaced across the range of 60 to 400 bases (60, 80, 100, 120, 140, 160, 180, 200, 225, 250, 275, 300, 325, 350, 375, and 400 bases) (Figure 4). These fragments have been labeled with a third fluorescent dye, carboxy-X-tetramethylrhodamine which allows placement of the marker in every lane. This provides detection of lane-to-lane variation in product migration in the gels which can then be corrected using the detection instrument software. Instrument software also allows comparison of migration of the known sizes of the allelic ladders with the unknown alleles in test samples, providing the most accurate determination of alleles possible.

Previously available size markers have been limited by the uneven spacing of fragments contained within the marker set, and by the different sequences contained within the different fragments of the marker set. These limitations can cause imprecision in defining sizes of unknowns, especially in regions which have a paucity of fragments, and may cause deviation from a linear sizing plot resulting from the varying sequences. Each of the 16 fragments of the marker described here contains the entire sequence of all smaller fragments in the set. That is to say, the small fragments are subsets of each larger fragment. This characteristic limits deviation from linearity in the measurements, while the even spacing provides increased precision.

Figure 5 illustrates simultaneous electrophoresis and detection of the GenePrint™ PowerPlex™ System with the Fluorescent Ladder size marker using the Hitachi FMBIO^® fluorescent scanner. Figure 6 illustrates the same combination detected with the ABI Prism™ 377 DNA Sequencer. By including the size marker in each lane, the number of lanes containing allelic ladder can be reduced to one or two, increasing the lanes available for samples, and generating very high throughput.

Preliminary development of population statistics for these loci, and their various multiplex combinations has been completed. Generation of these data included analysis of more than two hundred individuals from three major racial and ethnic groups present in the United States, i.e. African-Americans, Caucasian-Americans, and Hispanic-Americans. Analysis of Asian populations has not been completed at this time. The number of individuals tested, the heterozygosity, and the separate allele frequencies for each locus in each racial/ethnic group are displayed in Table 4. Alleles for each system are inherited in Mendelian fashion.

The commonly used statistical information for description of polymorphic systems employed in forensic and paternity laboratories has been determined for each fluorescent multiplex combination. Discrimination power is often measured as the probability of a match (Jones, 1972), i.e. the average number of individuals who would have to be surveyed to find a match with a randomly selected individual. Table 5 displays the probability of a match for each system and each racial/ethnic group. Note that the PowerPlex™ System alone provides matching probabilities which exceed 1 in 118,000,000 in each group. When the PowerPlex™ System and the FFFL multiplex are used in combination, these numbers range from 1 in 178 billion to approximately 1 in three trillion, many times the population of the earth.

A measure of discrimination often used in paternity analyses is the paternity index (PI) which is a means for presenting the genetic odds in favor of paternity given the genotypes for the mother, child, and alleged father (Brenner and Morris, 1989). The typical PIs for each fluorescent multiplex are displayed in Table 6. The PowerPlex™ system alone provides typical paternity indices exceeding 300 in each racial/ethnic group, enough to satisfy routine requirements for paternity determination. With the inclusion of the FFFL multiplex, the values exceed 5000 in all cases.

An alternative calculation used in paternity analyses is the power of exclusion (Brenner and Morris, 1989). This value, calculated for the eight-locus PowerPlex™ system exceeds 0.997 in all races tested (Table 6). In combination with the FFFL multiplex, the values exceed 0.9998, again demonstrating the usefulness of these two systems for paternity analyses as well as forensic determinations.

Our group has learned a number of important lessons during the development of STR multiplex systems. In particular, there are two examples worth noting of the importance regarding proper primer selection. First is the case of partial allele dropout, or allele ambiguity, which has been observed at the D7S820 locus.

Comparison in a variety of primers for amplification of the locus D7S820 has displayed variable results. When selected samples were amplified with the primers described by the Cooperative Human Linkage Center (CHLC, 1996), they appeared as homozygotes (Figure 7). Amplification of these same samples using primers described at the Sixth International Symposium on Human Identification in a poster by Fourney et al. (1996) produced a more enigmatic result, an apparent heterozygote with one allele displayed more strongly than the other (Figure 7).

Sequence analysis of the D7S820 locus in several individuals indicated a C to T base substitution is present in a small, but significant number of individuals (as much as 9% of African-Americans tested). This polymorphism is in the region which hybridizes with one primer in each of the previously reported pairs (Figure 9). Thus, it appears that the partial allele dropout occurring with these primers was due to imperfect hybridization with some, but not all, of the DNA templates. We have designed new primers for inclusion in the PowerPlex™ System which amplify a larger region of the D7S820 locus and accurately reveal the true nature of these heterozygous individuals (Figure 7, Figure 8).

This experience clearly demonstrates the importance of proper design of primer sequences and the necessity to test a large sample population for diversity. In this particular case, the African-American population shows the allele ambiguity most frequently than the Caucasian-American group.

A second case illustrating the importance of primer design relates to use of the newly available enzyme, AmpliTaq Gold (Perkin Elmer – Applied Biosystems, Foster City, CA). Reports in advertising

relating to this enzyme indicate that, compared to the standard Taq DNA Polymerase, it sometimes diminishes or eliminates artifact bands generated by improper interaction of primers with one another, with template, or with products of this combination during multiplex amplifications. Our own experience is that these artifacts are primarily a problem related to poor primer design, and that when primers are designed appropriately, artifact-free amplification products are produced with the standard Taq DNA Polymerase as well as the AmpliTaq Gold enzyme. Figure 10 illustrates sensitive artifact-free amplification of the PowerPlex™ System using each enzyme.

The need for highly discriminating polymorphic systems which can handle both low throughput and high throughput applications with convenience and flexibility can be met by combining the newly developed multiplex systems and fluorescent scanners described in this work. Matching probabilities exceeding 1 in 118,000,000 for an individual multiplex system have been achieved with the GenePrint™ PowerPlex™ System. Employing the Hitachi Software Engineering FMBIO^® fluorescent scanner allows analysis of dozens of gels per day as well as a convenience of bunching and shortening the time dedicated to gels when only two or three gels per day are required. When additional discrimination power is occasionally required for paternity analyses, the multiplex combination of PowerPlex™ and FFFL provide powers of exclusion which, in combination, exceed 0.9997 for each racial/ethnic group.

These STR multiplex systems offer the efficiency, reliability, and discrimination power required for database analysis, forensic science, and paternity determination applications.

We would like to thank Steve D. Creacy and Robert A. Bever for their collaboration on development of the population data described in this work.

Figure 1. GenePrint™ Fluorescein-Labeled STR Multiplex Systems. In each panel, six DNA samples have been amplified (lanes 1 - 6) and are shown with allelic ladders for the corresponding system (lanes labeled L). Panel A displays a two-color image of all eight STR systems which have been amplified simultaneously and detected using the Hitachi FMBIO^® fluorescent scanner. The amplified products of the fluorescein-labeled loci, D16S539, D7S820, D13S317, and D5S818, are shown in green, while the TMR-labeled loci, CSF1PO, TPOX, TH01, and vWA are shown in red. Panel B displays the 505nm scan which reveals the corresponding black and white image of the fluorescein-labeled loci, D16S539, D7S820, D13S317, and D5S818. Panel C displays the 625nm scan which reveals a black and white image of the TMR-labeled loci, CSF1PO, TPOX, TH01 and vWA. In Panels B and C, each allelic ladder is labeled to its right with the number of copies of the repeated sequence contained within its corresponding largest and smallest alleles. All materials were separated using a 4% polyacrylamide denaturing gel.

Figure 2. The GenePrint™ PowerPlex™ – Hitachi-Compatible System. Six DNA samples have been amplified (lanes 1 - 6) and are shown with allelic ladders for the corresponding system (lanes labeled L). Panel A displays a two-color image of all eight STR systems which have been amplified simultaneously and detected using the Hitachi FMBIO^® fluorescent scanner. The amplified products of the fluorescein-labeled loci, D16S539, D7S820, D13S317, and D5S818, are shown in green, while the TMR-labeled loci, CSF1PO, TPOX, TH01, and vWA are shown in red. Panel B displays the 505nm scan which reveals the corresponding black and white image of the fluorescein-labeled loci, D16S539, D7S820, D13S317, and D5S818. Panel C displays the 625nm scan which reveals a black and white image of the TMR-labeled loci, CSF1PO, TPOX, TH01, and vWA. In Panels B and C, each allelic ladder is labeled to its right with the number of copies of the repeated sequence contained within its corresponding largest and smallest alleles. All materials were separated using a 4% polyacrylamide denaturing gel.

Figure 3. The GenePrint™ PowerPlex™ – ABI-Compatible System. The electropherogram of a DNA sample co-amplified at eight loci is shown. The amplified products of the fluorescein-labeled loci, D16S539, D7S820, D13S317, and D5S818, are shown as blue peaks, while the TMR-labeled loci, CSF1PO, TPOX, TH01, and vWA are shown as black peaks. All materials were separated using a 4% polyacrylamide denaturing gel and detected with the ABI Prism™ 377 DNA Sequencer.

Figure 4. The GenePrint™ Fluorescent Ladder. The electropherogram from the ABI Prism™ 377 DNA Sequencer displays the 16 fragments contained within the newly developed fluorescent size marker. The carboxy-X-tetramethylrhodamine-labeled fragments appear in red with their respective sizes, in bases, listed above each fragment.

Figure 5. The GenePrint™ PowerPlex™ – ABI-Compatible System Illustrated with Fluorescent Ladder (Size Marker). The electropherogram of a DNA sample co-amplified at eight loci is shown. The amplified products of the fluorescein-labeled loci, D16S539, D7S820, D13S317, and D5S818, are shown as blue peaks, while the TMR-labeled loci, CSF1PO, TPOX, TH01, and vWA are shown as black peaks. Fragments of the Fluorescent Ladder (size marker) are illlustrated in red. All materials were separated using a 4% polyacrylamide denaturing gel and detected with the ABI Prism™ 377 DNA Sequencer.

Figure 6. The GenePrint™ PowerPlex™ – Hitachi-Compatible System Illustrated with Fluorescent Ladder (Size Marker). Eighteen DNA samples have been amplified (lanes 1-30) and are shown with allelic ladders for the corresponding systems (lanes labeled L) displayed in purple and green for the TMR-labeled loci, CSF1PO, TPOX, TH01, and vWA, and the fluorescein-labeled loci, D16S539, D7S820, D13S317, and D5S818, respectively. Fragments of the Fluorescent Ladder (size marker) are illustrated in orange. Numbers to the right of the figure indicate the sizes (in bases) of the respective fragments of the Fluorescent Ladder. All materials were separated using a 4% polyacrylamide denaturing gel and detected with the Hitachi Software Engineering FMBIO^® fluorescent scanner.

Figure 7. Effects of primer selection on product yield of the D7S820 locus. In each panel, lanes 1-10 contain the same DNA samples amplified with Primer Set 1 (CHLC, 1996), Primer Set 2 (Fourney et al., 1996), or the GenePrint™ Primers (this work), respectively, for the locus D7S820. Samples in lanes 1-5 display different relative yields of the two alleles present in each sample using Primer Sets 1 and 2, but more even yields with the GenePrint™ Primers. Samples in lanes 6-10 display relatively even amplification yields of the two products using any of the primer sets.

Figure 8. Quantitation of the primer selection effect on product yield of the D7S820 locus. Each panel displays a line tracing from the Hitachi FMBIO^® fluorescent scanner for the same two samples amplified with Primer Set 1 (CHLC, 1996), Primer Set 2 (Fourney et al., 1996) or the GenePrint™ Primers (this work), respectively, for the locus D7S820. The upper sample displays allele dropout for the lower allele using Primer Sets 1 and 2, but more even yields with the GenePrint™ Primers. The lower sample in each set displays relatively even amplification yields of the two products using any of the primer sets.

Figure 9. Display of sequence difference underlying the effects of primer selection. The region of the thick line indicates the position of the repeated sequence of the D7S820 locus in the context of surrounding DNA sequence, represented by the thin line extending to either side of the repeat. Primers described in the text are indicated by shorter lines with arrows representing the 3' terminus of each primer. The "C" or "T" identification indicates the sequence present on the positive strand of the genomic sequence or its corresponding primer. The relative peak heights of amplification products generated for each primer set for each of the genomic sequence options (See Figure 8) are illustrated to the right of the figure.

Figure 10. Comparison of the GenePrint™ PowerPlex™ amplified with Taq DNA Polymerase
or AmpliTaq™ Gold. The left and right lanes of each panel contain allelic ladders for the 8 PowerPlex™ STR loci. The remaining lanes contain amplified products of 10, 2, 1, 0.5, 0.2, and 0.0ng DNA templates, respectively. DNA templates illustrated in the left panel were amplified using AmpliTaq™ and the GenePrint™ STR Buffer, while those in the right panel were amplified using AmpliTaq™ Gold and its accompanying buffer.