J.W. Schumm, C.J. Sprecher, A.M. Lins, K.A. Micka, D.R. Rabbach, J.A. Taylor, Allan Tereba, and J.W. Bacher. Promega Corporation, 2800 Woods Hollow Rd., Madison, WI, 53711 USA.

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Especially high throughput can be achieved with the re-use of polyacrylamide gels (described here) in conjunction with the Hitachi FMBIO^® II Fluorescent Scanner. This approach minimizes the pouring of gels and simplifies the ability to separate amplified products of several hundred samples per day allowing several thousand locus calls per day with a single scanner instrument.

Also discussed in this presentation is the identification of a new class of loci which reveal less of the artifact known as stutter than previously described loci. This is achieved even for loci which display a high degree of polymorphism.

The GenePrint^TM PowerPlex^TM 1.1 System (Figure 1) was described at last year’s symposium (Schumm et al.). This system provides simultaneous amplification of eight STR loci which may be separated in a single gel lane as seen in Figure 1. A single primer for each of four primer pairs for loci D16S539, D7S820, D13S317, and D5S818 is labeled with fluorescein, and four others are labeled with tetramethyl-rhodamine (TMR) (CSF1PO, TPOX, TH01, and vWA). An additional size marker may be included in every gel lane to monitor potential lane-to-lane variations in electrophoretic migration of amplified materials.

The GenePrint^TM PowerPlex^TM 1.1 System and the GenePrint^TM FFFL Fluorescent STR System, a second multiplex system containing the loci FESFPS, F13A01, F13B, and LPL, were used to determine the genotypes of at least 200 individuals in African-American, Caucasian-American, and Hispanic-American populations, respectively, at each of the twelve loci.

In a one-locus exact test of Hardy-Weinberg Equilibrium (Table 3), only one significant deviation from the expectation of random inheritance of alleles (the locus D13S317 in the Caucasian-American population displays a value of 0.021, shown in bold in Table 1) was observed. From statistical variation in data analysis alone, we would expect, on average, to observe 1.8 values below 0.050 in the data set of 36 tests described here if there was Hardy-Weinberg equilibrium. Thus, the observed uniform distribution of the 36 p-values over the range 0 to 1 is the result expected if there are no departures from Hardy-Weinberg equilibrium at these 12 loci in these 3 databases.

One of the inconveniences of the current methods of STR analysis relates to the pouring of polyacrylamide gels and the related cleaning of glass plates. We have discovered that this can be minimized by re-use of poured plates. In fact, a single gel can be used at least six times over a one week period saving both time and expense. Figure 3 illustrates the use of the same gel to analyze the Fluorescent Ladder (CXR), 60-400 Bases size standard on repeated electrophoretic runs. Gels were subjected to electrophoresis, scanned, and then subjected to electrophoresis with reversed polarity to remove the original fragments. These warmed gels, were then loaded and subjected to electrophoresis in the forward direction again followed by scanning of the newly loaded materials. As seen in Figure 3, the gels maintain their ability to separate fragments precisely for at least six rounds of electrophoresis and reverse electrophoresis. The corresponding line traces also show no deterioration (data not shown). The complete protocol for this application is described in Figure 4.

One of the perennial goals of those developing STR loci has been the isolation, characterization, and development of systems which display a high degree of heterozygosity but reveal fewer artifact bands, such as the stutter bands so prevalent with loci such as vWA and FGA. After an extensive search of the human genome, we have identified a small number of these loci. Examples are displayed in Figure 5. While these systems are highly polymorphic in each population investigated, essentially no stutter artifacts are observed. We will continue to develop and describe these loci for inclusion into new multiplex systems.

We would like to thank Dr. Robert A. Bever (Bode Technologies, Sterling, VA) and Steven D. Creacy (Creacy Enterprises, Inc (Greensboro, NC) for assistance with the generation of population database results. We would also like to thank Dr. B.S. Weir, R.J. Morris, and J.D. Storey of the Program in Statistical Genetics at North Carolina State University for kindly performing some of the statistical analyses described in this work.

Schumm, J.W., Lins, A.M., Micka, K.A.., Sprecher, C.J., Rabbach, D.R., and Bacher, J.W. Fluorescent Detection of STR Multiplexes--Development of the GenePrint^TM PowerPlex^TM and FFFL Multiplexes for Forensic and Paternity Applications. Proceedings from the Seventh International Symposium on Human Identification, 1996, Promega Corporation, pp. 70-88.

Figure 1. DNA samples amplified using the GenePrint^TM PowerPlex^TM 1.1 System. Amplification products were separated by electrophoresis in a 4% denaturing polyacrylamide gel and visualized using the Hitachi FMBIO^® II Fluorescent Scanner. The image on the left simultaneously displays three scans of the same gel at 505nm (displayed in green), 585nm (displayed in red, and 650nm (displayed in blue), respectively. The blue fragments illustrate a subset of the Fluorescent Ladder (CXR), 60-400 bases, which acts as a size standard. The other three images are black and white representations of the 505nm, 585nm, and 650nm scans shown separately. The respective loci displayed in the 505nm and 585nm scans are listed. The sizes of fragments of the size marker, present in every lane of the gel, are listed to the left of the 650nm scan. Lanes labeled (L) contain the PowerPlex^TM Allelic Ladder mixture. Numbered lanes contain individual amplified DNA samples. The smallest and largest allele of each locus, defined by the number of repeat units contained within it, is labeled to the right of the allelic ladders.

Figure 2. The GenePrint^TM PowerPlex^TM 1.2 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 illustrated in red. All materials were separated using the ABI PRISM^TM 310 Genetic Analyzer.

Figure 3. Re--Use of Denaturing Polycrylamide Gels. A 4% denaturing polycrylamide gel was loaded with Fluorescent Ladder (size marker), and subjected to electrophoresis (Run 1).  The fragments were removed by reverse electrophoresis, the Fluorescent Ladder reloaded, and again subjected to forward electrophoresis (Run 2). This process was repeated twice more (Run 4) and again twice more (Run 6). Following each separation, fragments were detected using the Hitachi FMBIO^® II Flourescent Scanner.

Figure 5. New loci displaying high heterozygosity and low stutter artifact. Several arbitrarily selected individual DNA samples were amplified using fluorescently-labeled primers for new STR loci, separated by gel electrophoresis, and detected using the Hitachi FMBIO^® II Fluorescent Scanner.