Evaluation of five polymorphic microsatellite markers for typing DNA from decomposed human tissues--correlation between the size of the alleles and that of the template DNA.
For PCR-based genotyping using polymorphic microsatellite markers, DNA from decomposed postmortem human tissues was fractionated into six groups according to molecular size. The minimum required amounts of this degraded DNA, for detecting alleles at five microsatellite loci (ACTBP2, CMAG, HUMTH01, CYP19, and LPL) and one minisatellite locus (MCT118) were investigated respectively. The allele patterns were detected by electrophoresis of the PCR products on a 6%-denaturing polyacrylamide gel following silver staining. The detection of alleles for the loci with large allele size required more template DNA with higher molecular size than for that with small allele size. Amounts from 0.3 ng to 5 ng were needed for allele detection on genomic DNA from fresh blood. When the decomposed DNA mixture was used as the template, approximately ten times the amount of genomic DNA was required to detect alleles at the three loci of LPL, CYP19 and HUMTH01, while 24 to 67 times was required for the loci, CMAG, ACTBP2 and MCT118. It was demonstrated that a minimum molecular, size and amount of template DNA was needed for amplifying alleles of the six loci, and degraded DNA less than minimum size in the samples would prevent the detection of the loci which have large allele size.
Takahashi
M
M
Department of Legal Medicine, Dokkyo University School of Medicine, Tochigi, Japan.
Kato
Y
Y
Mukoyama
H
H
Kanaya
H
H
Kamiyama
S
S
D000483:Alleles; D004247:DNA / Q000032:analysis*; D016172:DNA Fingerprinting / Q000379:methods*; D015894:Genome, Human; D005838:Genotype; D006801:Humans; D018895:Microsatellite Repeats / Q000235:genetics*; D008970:Molecular Weight; D010316:Particle Size; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D011180:Postmortem Changes; D015203:Reproducibility of Results
D004247:DNA