Electrophoresis zyxwvutsrqponmlkj 1996, 17, 1505-1511 DNA Typing of a PCR-amplified D1SBO/amelogenin multiplex 1505 z Alice R. Isenberg’ Bruce R. McCord’ Barbara W. Koons‘ Bruce Budowle’ Ralph 0. Allen’ ‘Department of Chemistry, University of Virginia, Charlottesville, VA, USA ’FBI Laboratory, Forensic Science Research Unit, Quantico, VA, USA DNA Typing of a polymerase chain reaction amplified DlS8O/amelogenin multiplex using capillary electrophoresis and a mixed entangled polymer matrix In this study, a technique was developed to separate by capillary electropho- resis (CE) the widely varying DNA fragment sizes produced by a multiplex polymerase chain reaction (PCR) amplification of the loci DlS80 and amelo- genin. Experiments were performed to analyze different buffer systems and obtain optimal resolution for the separation. A matrix composed of two diffe- rent molecular weights of the same polymer was constructed to separate the DNA fragments with baseline resolution, and a cubic spline fit was used to estimate the size of DNA fragments over 350 base pairs. Over 100 samples were examined to demonstrate the rapid, robust and precise characteristics of this CE system. An average relative standard deviation of 0.3% was obtained for the sizing of the D1S80 alleles in these samples. DNA from mixed body fluid samples, samples subjected to environmental insult, and DIS80 sequence variants were also typed successfully. These results demonstrate that CE is a viable method for analysis of DlS80 and amelogenin forensic DNA samples. 1 Introduction Variable number tandem repeat (VNTR) loci are used in human identity testing to discriminate between individ- uals at the DNA level [I]. One of the most comprehensi- vely studied VNTRs is the DlS80 locus [l-71. DlS80 is located on chromosome 1 of the human genome and contains a 16 base pair (bp) repeat [8]. DlS80 alleles range in size from approximately 369 bp to greater than 800 bp [9] and can be amplified easily via the poly- merase chain reaction (PCR). The DlS80 system has been used widely by the forensic community for identity testing [l-71. Another forensic marker of interest is the amelogenin locus [lo]. The typing of the amelogenin gene enables gender determination of the source of a DNA sample. This gene is carried on the sex chromo- somes, and the fragment of interest is six base pairs longer in the Y chromosome than in the X chromosome in humans [lo]. Following amplification, differentiation between females and males is possible by determining if one or both of the fragments, respectively, is present. It has been demonstrated previously that DlS80 and ame- logenin can be amplified by PCR simultaneously as a “multiplex” [ll]. Since the amelogenin PCR product is a different size than that of the DlS80 PCR product, elec- trophoretic separation of the multiplex is relatively simple. Slab gel electrophoresis has been used to separate PCR- amplified fragments of DNA on a routine basis. How- ever, capillary electrophoresis (CE) offers several advan- tages over slab gel electrophoresis including automation, increased precision, and the ability to quantitate results. Correspondence: Dr. Bruce R. McCord, FBI Academy, Forensic Science Research Unit, Building 12, Quantico, VA 22135, USA (Tel: +703-640-1539; Fax: +703-640-1394; E-mail: 103454.2264@compu- serve.com) Nonstandard abbreviations: HEC, hydroxyethylcellulose; dNTP, deoxy- ribonucleoside-5‘-triphosphate; YO-PRO-1, oxazole yellow Keywords: DlS80 zyxwvutsrqpo / Amelogenin / Capillary electrophoresis / Laser- induced fluorescence / Polymerase chain reaction / Forensic For separating DNA fragments, an aqueous solution of polymers is used to form a transient mesh in the solu- tion, which enables separation of DNA fragments according to size [12, 131. The high sensitivity of laser- induced fluorescence (LIF) allows PCR-amplified sam- ples to be analyzed directly following a simple dilution step [14]. The DNA can be derivatized for detection by using intercalating dyes added directly to the buffer [15, 351. The fluorescence of intercalating dyes is enhanced up to 1000 times through the intercalation process, with minimal background fluorescence [ 161. Additionally, the intercalating dye molecules can improve overall resolu- tion by unraveling and elongating the DNA molecule, minimizing conformational effects, and aiding in the interaction between the DNA and the polymer [17]. In this laboratory, we have developed procedures for the precise analysis of PCR-amplified DNA from 150-300 bp [14, 181 using this technique. Because of run-to-run variation in buffer viscosity, voltage, and temperature in CE, precise measurements can be made only through reference to internal stand- ards [19-201. Previous results in this laboratory have demonstrated peak migration time precision of below 0.1 O/o relative standard deviation (RSD) using this approach [21]. In addition, precise estimates of DNA size can be produced by interpolation of the PCR product between two internal standards 114, 181 because DNA migrates linearly with respect to fragment length below 400 bp [22]. Above 400 bp, DNA does not migrate lin- early with respect to fragment size, and an alternative method of sizing must be implemented. The wide varia- tion in fragment sizes (200-800 bp) produced by the DlS8O/amelogenin amplification required us to modify our experimental procedures. By mixing two polymers of varying length in the separation matrix, baseline resolu- tion of all alleles in the DlS80/amelogenin mulitplex was obtained. DNA size was determined by using a non- linear curve-fitting technique employing internal stand- ards and a DNA allelic ladder. All results were compared with slab gel results to ascertain the accuracy of the CE protocol. zyxw 0 VCH Verlagsgesellschaft mbH, 69451 Weinheim, 1996 0173-0835/96/0909-1505 $10.00+.25/0