DNA Typing of vWA Locus from Dental Pulp using Capillary Gel Electrophoresis
H. Tsutsumi, H.
Ohtaguro, R. Mukoyama, and T. Komuro
Department of Legal Medicine, Nihon University School of Dentistry, Tokyo, Japan
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VWA typing of DNA extracted from dental pulp was conducted by capillary polyacrylamide gel electrophoresis (CGE).
MATERIALS AND METHODS
Dental Pulp DNA
DNA samples were obtained from teeth stored in room for 1-23 years, by using phenol/chloroform extraction and precipitation with ethanol.
Amplification of vWA Locus
The quantity of template DNA was used 10 ng. Amplification of vWA locus was performed by the PCR method with GenePrintÔ STR kit (Promega) according to manufacture’s protocol.
Preparation of vWA Ladder Marker
Each allele of the vWA locus was detected by denaturing polyacrylamide gel electrophoresis (PAGE) and silver staining. DNA refined from the detected DNA band was used as template DNA to amplify again by the PCR method. These PCR products were used as ladder marker for vWA typing.
CGE
Quanta 4000 (Waters) was used as the CGE equipment. The capillary column of an inner diameter of
75 mm and 35cm length was filled with PAG(T=5%, C=0.5%). All samples were introduced into the column by electromigration (260 V/cm for 80 sec.). Electrophoresis was carried out at 240 to 310 V/cm for 40 minutes, using the buffer for electrophoresis containing 0.1M of tris-borate acid and 7M of urea. The vWA loci were detected by 254nm UV absorption.
RESULTS AND DISCUSSION
Influence of Electrophoesis Voltage on Migration Time
Two peaks appeared in about 20 minutes when electrophoresis was carried out at 310 V/cm using alleles 14 and 18 of vWA ladder marker. They appeared in about 28 minutes in the case of electrophoresis at 260 V/cm, and after 30 minutes in the case of electrophoresis at 240 V/cm. Namely, detection of each of allele 14 and 18 was delayed with reducing voltage and migration time difference tended to increases.
vWA Typing from Dental Pulp
1. vWA typing based on migration time
CGE of known alleles was carried out, and a calibration curve was prepared for vWA tying, using the electrophoresis startup time and allele migration time as reference point. Based on this calibration curve, other known alleles were subjected to electrophoresis, but correct determination of alleles failed. This is considered as follows: Alleles detection time was not constant, despite the same electrophoresis condition.
2. vWA typing based on migration time of primer
Known alleles were subject to CGE, and a calibration curve was prepared, using the primer detecting time and allele migration time as reference points. Based on this calibration curve other known alleles were subjected to electrophoresis, and this allowed correct determination of alleles. Then 20 samples of dental pulp were subjected to electrophoresis. All of the typing of vWA by CGE were also confirmed with denaturing PAGE.
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