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Anomalous Amplification of the Amelogenin Locus Typed by AmpF/STR Profiler Plus Amplification Kit, by Shewale, Richey, and Sinha (Forensic Science Communications, October 2000)

Anomalous Amplification of the Amelogenin Locus Typed by AmpF/STR Profiler Plus Amplification Kit, by Shewale, Richey, and Sinha (Forensic Science Communications, October 2000)

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October 2000 - Volume 2 - Number 4

Anomalous Amplification of the Amelogenin Locus
Typed by AmpFLSTR® Profiler Plus™ Amplification Kit

Jaiprakash G. Shewale, Stephen L. Richey,
and Sudhir K. Sinha

ReliaGene Technologies, Incorporated
New Orleans, Louisiana

Abstract | Introduction | Materials and Methods
Results and Discussion | References


Abstract

Among the 7,220 male samples analyzed, the X chromosome-specific allele at the Amelogenin locus was not amplified in three samples using the AmpFLSTR® Profiler Plus™ amplification kit. However, the X chromosome-specific allele in these samples was amplified using the GenePrint™ Sex Identification System–Amelogenin amplification kit. The most probable explanation for this anomalous phenomenon is that these samples had a mutation on the X chromosome within the primer-binding site for the specific primer provided in the AmpFLSTR® Profiler Plus™ amplification kit.

Introduction

Polymerase chain reaction (PCR)-based human identification systems have become the method of choice for forensic DNA casework and felon databases in recent years (Budowle et al. 1998; Edwards et al. 1992; Fregeau et al. 1993). These PCR systems have made possible the analysis of samples containing low quantities of DNA, such as stains from body fluids and cigarette butts, and ancient or highly degraded samples. Many of these systems exploit highly polymorphic short tandem repeat (STR) sequences, which offer an extremely high degree of discrimination between individuals within a population (Fregeau et al. 1993; Kimpton et al. 1993; Lins et al. 1998; Urquhart et al. 1995). The size of STR fragments is generally in the range of hundreds of base pairs as compared to thousands of base pairs found in restricted fragment length polymorphism (RFLP). Multiplex amplification systems such as AmpFLSTR® Profiler Plus™, AmpFLSTR® COfiler™, GenePrint™, PowerPlex™ 1.1 System, CTTv Quadriplex, and FFFL Quadriplex are commercially available. Some multiplex amplification systems contain sex-determinant markers.

Amelogenin is not an STR locus, but it produces X and Y chromosome-specific PCR products of different sizes. Coamplification of Amelogenin with STR loci provides a combined gender–identity test (Sullivan et al. 1993). Multiplexing the Amelogenin with other STR loci has been useful in forensic analysis for characterizing the mixture of male and female DNA.

During the analysis of reference-blood samples by using the AmpFLSTR® Profiler Plus™ amplification kit, the allele for the X chromosome at the Amelogenin locus was not amplified in a few samples. The interpretation, confirmation, and consequences of the results are discussed.

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Materials and Methods

Genomic DNA was extracted from bloodstain cards, whole blood, or both using the Chelex-extraction method (Perkin-Elmer 1991). PCR was performed by using AmpFLSTR® Profiler Plus™ PCR amplification kit (Perkin-Elmer Applied Biosystems, Foster City, California) and GenePrint™ Sex Identification System–Amelogenin (Promega Corporation, Madison, Wisconsin).

PCR reactions using Perkin-Elmer's kits: PCR reaction mixes were prepared as per the manufacturer's guidelines (Perkin-Elmer 1991). Each reaction for amplification using the AmpFLSTR® Profiler Plus™ kit contained 20.0 µL AmpFLSTR® Reaction Mixture, 10.0 µL AmpFLSTR® Primer Set, 2.0 µL of extracted DNA (2 ng), 17.0 µL of sterile water, and 1.0 µL of AmpliTaq Gold DNA Polymerase (5 U/µL). The amplification reaction was carried out in the GeneAmp® PCR System 9700 (Perkin-Elmer Applied Biosystems, Foster City, California). The first cycle, an enzyme-activation step, was a 95-degrees Celsius hold for 11 minutes. The cycling parameters were 94 degrees Celsius for one minute, 59 degrees Celsius for one minute, and 72 degrees Celsius for one minute, and the reaction completed 28 cycles. The final extension was at 60 degrees Celsius for 45 minutes, followed by a 4-degrees Celsius soak. Amplified products, after denaturation (95 degrees Celsius, three minutes), were analyzed using the ABI Prism® 310 Genetic Analyzer (Perkin-Elmer Applied Biosystems, Foster City, California) using performance optimized polymer (POP) four polymer and standard instrumental conditions as specified by the manufacturer. The data were analyzed (Perkin-Elmer 1998) using GeneScan™ and Genotyper™ software (Perkin-Elmer Applied Biosystems, Foster City, California).

PCR reactions using Promega's kits: The second method for PCR amplification used the GenePrint™ Sex Identification System–Amelogenin (Promega Corporation, Madison, Wisconsin). The reaction mixture contained 13.8 µL sterile-deionized water, 1.0 µL bovine serum albumin (10X), 2.5 µL STR buffer (10X), 2.5 µL Amelogenin Primer pair mix, 0.2 µL AmpliTaq® DNA Polymerase (5 U/µL), and 5.0 µL of extracted DNA (2 ng; Promega Corporation 1997). Amplification was performed in the DNA Thermal Cycler 480 (Perkin-Elmer Applied Biosystems, Foster City, California). PCR consisted of one cycle at 96 degrees Celsius for two minutes, followed by 10 cycles at 94 degrees Celsius for one minute, 60 degrees Celsius for one minute, 70 degrees Celsius for 1.5 minutes, and an additional 20 cycles at 90 degrees Celsius for one minute, 60 degrees Celsius for one minute, 70 degrees Celsius for 1.5 minutes. An extension cycle of 60 degrees Celsius for 30 minutes completed the reaction. Amplified products were electrophoresed on a polyacrylamide gel and analyzed (Promega Corporation 1997) using the Genomyx SC genetic scanner (Genomyx Corporation, Foster City, California).

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Results and Discussion

A total of 7,600 reference-blood samples from multiple populations was analyzed using the AmpFLSTR® Profiler Plus™ amplification kit. Of these, 7,220 samples were from male donors. The allele profile for all female samples at Amelogenin locus was X, X. The allele profile for male samples at Amelogenin locus was X, Y except for three samples (designated as Samples 1, 3, and 4), wherein only the Y allele was observed. The Genotyper™ profiles of these three samples are presented in Figure 1.

The Samples 1, 3, and 4 showed amplification of one allele at the Y chromosome at the Amelogenin locus, which is anomalous. The amplification of positive and negative controls provided expected results (data not shown). It appears that the X chromosome-specific allele was not amplified. The presence of the X chromosome-specific allele in coamplified Sample 2 eliminated the possibility of PCR errors as a contributing factor. The results were reproducible. The three samples (Samples 1, 3, and 4), which exhibited anomalous results, and Sample 2, which exhibited amplification of both X and Y alleles, were amplified using the GenePrint™ Sex Identification System–Amelogenin amplification kit to find out whether or not X dropout occurred. This kit was selected because of its different primer set. The results are presented in Figure 2. The X chromosome-specific allele was amplified in all samples (Samples 1, 2, 3, and 4), and the allele call for Amelogenin locus was X, Y, as expected.


Graphic image showing the allele profiles of amplified genetic samples
 

 
Figure 1
Genotyper profile at Amelogenin
locus of the samples amplified
using AmpFLSTR® Profiler Plus™ amplification kit. The samples were analyzed on an ABI Prism® 310
Genetic Analyzer.


Graphic image showing the amplification of X and Y chromosome-specific alleles


Figure 2
Gel image of the samples amplified
using GenePrint™ Sex Identification
System–Amelogenin amplification kit.
The electrophoresed gel was analyzed
on Genomyx SC gel scanner. The top
band corresponds to Y chromosome-
specific PCR product, and the bottom
band corresponds to X chromosome-
specific PCR product. L=allelic ladder; P=positive control; N=negative control;
1=Sample 1; 2=Sample 2; 3=Sample 3;
and 4 = Sample 4.

The sizes of the X and Y chromosome-specific amplicons vary among manufacturers of the amplification kit because of variation in the primer sequence. The sizes of X and Y chromosome-specific alleles produced by the GenePrint™ Sex Identification System–Amelogenin amplification kit are 212 and 218 base pairs. The sizes from the AmpFLSTR® Profiler Plus™ amplification kit and analyzed by using ABI Prism® 310 Genetic Analyzer are 103 and 109 base pairs (Perkin-Elmer 1991, Promega Corporation 1997).

The female samples have two Xs, and if one of these did not amplify, the genotype would still be correct. In the male samples, nonamplification of X would raise a flag but would not lead to incorrect sex typing because Y is amplified. However, nonamplification of the Y would lead to wrong sex determination if the other information of the case is not considered. The absence of X chromosome-specific allele products using AmpFLSTR® Profiler Plus™ amplification kit is a rare phenomenon, which was observed in three samples of 7,220 individuals tested from a multiple population. However, amplification of the Y chromosome-specific allele at this locus was achieved in the same amplification reaction. Further, amplification of both X and Y chromosome-specific alleles was obtained, for these samples, using GenePrint™ Sex Identification System–Amelogenin amplification kit. The possible explanation for this anomalous phenomenon is that Samples 1, 3, and 4 had a mutation on the X chromosome within the primer-binding site for the specific primer provided in AmpFLSTR® Profiler Plus™ amplification kit. It appears that the primer-binding site for primers of AmpFLSTR® Profiler Plus™ amplification kit is located at or near a region prone to mutation.

Although this event was observed only in the X chromosome in male samples, it does not preclude the possibility of a similar mutation being present in the Y chromosome or other STR locus. In forensic casework when the sex of evidentiary samples is unknown, a careful interpretation of data is necessary if this type of anomaly is observed. As stated earlier, different possibilities such as no amplification of Amelogenin, anomalous amplification of either X or Y is possible. In rape cases, when the presence of semen, sperm, or both is identified, a differential extraction of the sample is done to separate the sperm from epithelial cell DNA. If the female fraction is consistent with the female victim (i.e., X product) and the male fraction shows only X (i.e., no Y product), a flag would be raised suggesting no Y amplification. Such anomalous amplifications that result from mutation at the primer-binding site may be investigated by using another multiplex or singleplex system that features primers with alternative sequences.

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References

Budowle, B., Moretti, T. R., Niezgoda, S. J., and Brown, B. L. CODIS and PCR-based short tandem repeat loci: Law enforcement tools. In: Second European Symposium on Human Identification 1998. Promega Corporation, Madison, Wisconsin, 1998, pp. 73–88.

Edwards, A., Hammond, H. A., Lin, J., Caskey, C. T., and Chakraborty, R. Genetic variation at five trimetric and tetrametric tandem repeat loci in four human population groups, Genomics (1992) 12:241–253.

Fregeau, C. J. and Fourney, R. M. DNA typing with fluoroscently tagged short tandem repeats: A sensitive and accurate approach to human identification, Biotechniques (1993) 15:100–119.

Kimpton, C. P., Gill, P., Walton, A., Milligan, E. S., and Adams, M. Automated DNA profiling employing multiplex amplification of short tandem repeat loci, PCR Methods and Applications (1993) 3:13–22.

Lins, A. M., Micka, K. A., Sprecher, C. J., Taylor, J. A., Bachner, J. W., Rabbach, D. R., Bever, R. A., Creacy, S. D., and Schumm, J. W. Development and population study of an eight-locus short tandom repeat (STR) multiplex system, Journal of Forensic Sciences (1998) 43:1168–1180.

Perkin-Elmer Applied Biosystems. AmpFLSTR® Profiler Plus™ PCR Amplification Kit, User's Manual. Perkin-Elmer Corporation, Foster City, California,1991.

Perkin-Elmer Applied Biosystems. ABI Prism™ Genetic Analyzer, User's Manual. Perkin-Elmer Corporation, Foster City, California,1998.

Promega Corporation. GenePrint™ Fluorescent STR Systems, Technical Manual. Promega Corporation, Madison, Wisconsin, 1997.

Sullivan, K. M., Mannucci, A., Kimpton, C. P., and Gill, P. A rapid and quantitative DNA sex test: Fluorescence-based PCR analysis of X-Y homologous gene Amelogenin, Biotechniques (1993) 15:636–641.

Urquhart, A., Oldroyd, N. J., Kimpton, C. P., and Gill, P. Highly discriminating heptaples short tandem repeat PCR system for forensic identification, Biotechniques (1995) 18:116–121.

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