Coloning of the variable region of D8S210 locus and its application to the forensic individual identification for Korean population / 대한법의학회지

ABSTRACT

A multi-locus probe. called pHK-450. detecting multiple variable DNA fragments, was constructed by cloning of the 450 bp fragments into pCR(TM) vector, which could be obtained from amplification of the variable region of the D8S210 locus of human genome with a primer set of UF1. UR1, and UR2. The sequence data shows that the variable region is entirely G-C rich on one strand consisting of tetranucleotide repeat unit (GGAA)n at both margins and internally diversed regions with simple irregular repeat units of GGAA, GGAG, or GGGAA. The genetic complexixty of the resulting individual-specific DNA patterns was investigated by studying a Korean pedigree(both parents and 10 children). HaeIII and HinfI-generated DNA fragments are inherited to their children in a Mendelian manner with segregation ratio of 11.05 and 11.14. respectively. In the HinfI-digested genomic DNAs, the segregation of up to 25 variable DNA fragments from both parents could be analysed in a single sibship. Most of these variable DNA fragments detected by pHK-450 probe are not paired as allele, which suggests that they may be derived from 23 heterozygous loci. In the HaeIII-digested genomic DNA, 26 variable DNA fragments are segregated from both parents to 10 children. Excluding one allelic and three linked DNA fragments, 22 out of 26 scored DNA fragments may be derived from 23 hetrozygous loci. To determine the variability of DNA fragments, DNA samples from 113 unrelated Korean were digested with HinfI and hybridized using pHK-450 probe. Pairwise comparisons of individual variable DNA fragments show that the polymorphic patterns were highly specific to an individual. The mean probaility(X) that fragments in A individual is present in B individual is 0.0337. From the degree of maxiaml sharing, band approximate estimates of mean allele frequency and homozygosity is calculated being 0.187 and the mean heterozygossity is 89.69%. For pHK-450 probe, the probability that all 10 resolved DNA fragments in the 5-30 kb size range in an individual A are also present in a second unrelated individual B is (0.337)(10) or 7.1 X 10(-6).The above results suggest that the Muiti-locus probe, pHK-450, is suitable for rapid polymorphic marker generation and can be applied to individual identification in forensic medicine