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Application and Utilization of STR Multiplexes for Parentage AnalysisSteve D. Creacy, Charles M. Kelly and Robert A. Bever
The human genome contains hundreds of loci which contain tetrameric repeats of varying lengths (1,2). These short tandem repeat loci (STR) have been utilized for use in forensic and paternity case work (3,4). Last year we reported on the validation of five of the STR loci (CSF1PO, TPOX, TH01, vWA [formerly vWFA31], and FESFPS) for the use in paternity casework (5). This report describes the validation of three more loci: F13A01, F13B, and LPL (6,7,8). The systems were validated by determining the frequencies of heterozygotes in the population, allelic frequencies, and observed powers of exclusion for each of the loci. Two hundred exclusionary paternity cases of each racial group were used for determining the allele frequencies, heterozygosity values and powers of exclusion for the three STR loci. The allele frequencies were determined by analyzing the mothers and alleged fathers of these exclusionary cases. These exclusionary cases were previously analyzed by RFLP analysis using the loci D10S28, D2S44, D4S139, D17S26 and D1S339. DNA was isolated from whole blood using a modified non-organic extraction technique (9) and from buccal cells by phenol chloroform isolation (5). DNA was quantitated by a standard fluorescent technique (10) using a microplate fluorometer (Cambridge Technology, Inc.). Amplification for loci F13A01, F13B, and LPL was completed with reagents and protocols provided by Promega Corporation's GenePrint™ STR Kit DC6000 and DC6010. These procedures are described in the STR Systems GenePrint™ Technical Manual (Promega Corporation, Madison, WI). However, the procedures were slightly modified for use with the Biotherm III BioOven. A master mix containing the STR multiplex primers, 10X STR buffer, sterile water, and Taq DNA Polymerase (.01 units/ml, Perkin Elmer) is mixed with genomic DNA on ice or a cold block. The final reaction volume is 25 µl containing 10-50ng of DNA. DNA was amplified in a Biotherm III BioOven. Typical amplification conditions for the multiplex (F13A01, F13B, and LPL) consisted of a hot soak at 95ºC for 3 minutes, 30 cycles of 94ºC for 1 minute, 60ºC 1 minute and 70ºC for 1.5 minutes, followed by a final extension at 72ºC for 5 minutes. Positive control K562 DNA and a negative control were run on each batch of amplified DNA. Detection of Amplified Products Amplified DNA product was separated by polyacrylamide gel electrophoresis in denaturing conditions as described in the technical manual using a Gibco/BRL sequencing apparatus Model SA 32. DNA was heated to 95ºC for 2 minutes, then chilled on ice prior to loading 3 µl of amplified product or 3 µl of allelic ladder onto the gel. Electrophoresis was performed at 40W in 0.5X TBE (Tris Borate Electrophoresis) Buffer for 1.5 hours. DNA was detected using the fluorescent stain SYBR Green II (1:10,000 dilution of stock-FMC) and visualized on the Molecular Dynamics Fluorimager at low resolution. Amplified product was analyzed by comparing the specimen amplified DNA migration with the amplified STR allelic ladder. All specimens were analyzed twice independently. Allelic frequencies of F13A01, F13B, and LPL are reported in Figures 1-3. Allelic frequencies for CSF1PO, TPOX, TH01, vWA, and FESFPS have been previously reported (5). Heterozygosity values, theoretical power of exclusion, observed power of exclusion, and median PI are reported in Table I. The observed combined power of exclusion values of Triplex I (CSF1PO, TPOX and TH01) Triplex II (F13A01, F13B, and LPL) , Duplex I (FESFPS, vWA) and D1S80 are summarized in Table 2. The primary purpose of parentage testing is to exclude the falsely accused man. The ability of a genetic system or battery of genetic systems to exclude the falsely accused man is expressed as the power of exclusion. The American Association of Blood Banks Standards for Parentage Testing (11) has established an average power of exclusion of 95% to be the minimum standard for parentage testing. An average power of exclusion of one STR system is approximately 50%. Therefore, a battery of six STR systems or three STR systems and D1S80 meets this minimum standard set by the AABB. In some cases using a six STR system test, paternity is not resolved with two exclusions or a paternity index of 100 or a specific probability of exclusion of 95%. This is due to the presence of common alleles. Therefore, a battery of eight STR loci are initially tested for typical paternity casework. By testing eight STR loci an average power of exclusion greater than 99.5 % can be obtained. The eight-system STR test results in resolving more than 99% of the casework with two exclusions or a paternity index of greater than 100. If additional testing is needed to resolve the case, then the amplified systems D1S80, CYAR04, or D19S253 (3,12) are utilized. For the rare cases involving single genetic inconsistencies between the alleged father and child, RFLP testing and/or HLA testing is utilized in conjunction with the STR analysis. In conclusion, the analysis of STR loci to resolve paternity casework has been shown to be a reliable and useful technology. Enough STR loci are commercially available to resolve paternity casework by solely utilizing these loci and provide an average power of exclusion greater than 99.5% with paternity indices greater than one hundred. The authors thank Bruce Boeko, Jing Ming Liu and Helen Lawrence for technical/laboratory assistance, and Ruth Tutterow for editing assistance.
Table 1. Power of Exclusion and Paternity Index: Consistency Checks
h = heterozygosity Table 2. Varying Battery of Tests: Observed Power of Exclusion
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