Sex Determination from Dental Pulp using Capillary Gel Electrophoresis of Amelogenin Locus
T. Komuro, M. Nakamura, H. Tsutsumi,
and Y. Morita
Department of Legal Medicine, Nihon University School of Dentistry, Japan
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Sex of DNA extracted from dental pulp was determined by capillary gel electrophoresis (CGE) with amelogenin locus as a marker.
MATERIALS AND METHODS
Dental Pulp DNA
DNA was extracted by phenol/chloroform method from aged dental pulp obtained from teeth extracted from 10 males and 10 females and stored at room temperature for 1 year.
Amplification of Amelogenin Locus
The X-chromosome-specific alphoid repeat sequence 212 bp (X locus) and Y-chromosome-specific alphoid repeat sequence 218 bp (Y locus) were amplified by the PCR method according to protocol of GenePrintÔ Sex Identification System-Amelogenin kit (Promega) with dental pulp DNA 10ng as a template DNA.
Preparation of X- and Y-locus Standard Samples
The X and Y loci amplified by the PCR method were detected by polyacrylamide gel electrophoresis (PAGE) and silver staining. Then DNA was refined from the detected DNA band. It was used as a template DNA to amplify the X and Y loci again by the PCR method. These PCR products were used as standard samples.
CGE
Quanta 4000 (Waters) was used as the CGE equipment. The capillary column having an inner diameter of 75 mm and a length of 35cm was filled with PAG (T=8%, C=O%). After STR 2X Loading Solution was added to the PCR product of amelogenin locus in all samples, these were introduced into the column by electromigration (200 v/cm for 80 sec.) Electrophoresis was carried out at 260 v/cm for 55 minutes, using the buffer for electrophoresis containing 0.1M of tris-borate acid and 7M of urea. X and Y loci were detected by 254nm UV absorption.
RESULT AND DISCUSSION
Identification of Each Peak in Electropherogram
Analysis of the samples by CGE has resulted in detection of the peaks of dNTP, bromphenol blue and xylene cyanol as well as the peaks of X and Y loci. So each reagent was independently subjected to CGE, each peak was correctly identified.
Verification of X and Y Loci
The male sample was analyzed by CGE, and two peaks appeared. Standard X- or Y-locus sample was added to this sample to carry out CGE. The result was that forward peak and backward peak were each amplified; thus, two peaks were verified to be X locus and Y locus, respectively.
Furthermore, the female sample was analyzed by CGE, and one peak appeared. Standard X- or Y-locus sample was added to this sample to carry out CGE. The result was that the peak was amplified when the standard X-locus sample was added, whereas two peaks appeared when the standard Y-locus sample was added. The peak of the sample was verified to be X locus.
Sex Determination from Dental Pulp
CGE was conducted for 10 male samples and 10 female ones. Two peaks were verified in the male sample, while one peak was verified in the female sample. Sex was correctly determined in all samples.
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