Identification of individuals mtDNA typing Procedures and interpretation of results

Due to maternal inheritance and the very low rate of recombination, all copies of mtDNA are generally identical (homoplasmic). In other words, the mtDNA genome is haploid and its sequence is treated as a single locus (haplotype). Thus, two hypervariable regions (HV-I and HV-II) of mtDNA can be determined by direct sequencing after PCR-amplification of the regions. Even for heteroplasmic mtDNA containing two mtDNA types, the sequences can often be determined by direct sequencing because there are usually only one or at most two base differences between the two types.

Mitochondrial DNA typing starts with the extraction of total genomic DNA from samples, and then PCR-amplification is performed on HV-I and HV-II. Forensic investigators thought that the size of each region (-400 bp) was too large to amplify simply by using one primer that targets the entire HV-1 region and another primer that targets the entire HV-II region. As it is difficult to amplify large fragment sizes in aged or decomposed samples, they reasoned that reducing fragment sizes in such samples could allow for PCR-amplification to be performed. They therefore targeted amplicons -250 bp in length, including the primer binding sites, with a total of four overlapping primer sets for HV-I and HV-II (Figure 8.2) (Stoneking et al., 1991; Wilson et al., 1995). The PCR-amplified products were then sequenced using a fluorescent automated sequencing system, and the sequencing information was confirmed by analysis of both forward and reverse DNA strands. The sequence of the unknown (evidence) sample was displayed as the L-strand sequence, and nucleotide differences between the evidence sample and CRS (rCRS) were noted. For example, an mtDNA type of '263G, 315.1C' indicates that an adenine (A) at position 263 in the CRS was substituted by a guanine residue (G), that there was an insertion of cytosine (C) between position 315 and 316 and that the remaining sequence of this mtDNA type was the same as that of the CRS.

In order to evaluate the sequencing results from evidence and reference samples, useful interpretation guidelines have been provided by the DNA Commission of the International Society for Forensic Genetics (Carracedo et al., 2000) as follows:

576 16024

L172 L29 L16159 L15997 <- - <- -

11-ยป K1 A n/ip

Was this article helpful?

0 0

Post a comment