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Figure 8.3 Schematic representation of the human mitochondrial cyt b gene and the amplification strategy for human identification. (a) Positions and orientations of the primers are indicated by arrows; (■■■■■) the entire cyt b gene amplified by the LJ4/34/HJ5863 primer pair; the regions targeted for human-specific amplification using the L15674/H15782 primer pair. (b) Comparison of human and various animal nucleotide sequences of regions targeted by primers L15674 and H15782. GenBank accession numbers are given in parentheses. Reprinted from Matsuda et al. (2005) with permission from Elsevier

Figure 8.4 Amplification products of the primary and nested PCR. Amplified products were electrophoresed on 3% agarose gel and stained with ethidium bromide. (a) Primary PCR with outer primers (L14734/H15863): M, HaeIII digest marker (TaKaRa); lane 1, amplified product from human DNA; lane 2, chimpanzee; lane 3, gorilla; lane 4, Japanese monkey; lane 5, crab-eating monkey; lane 6, pig; lane 7, cow; lane 8, dog; lane 9, goat; lane 10, chicken; lane 11, rat; lane 12, tuna; NC, negative control (no template DNA was added). (b) Nested PCR with inner primers (L15674/H15782): PCR products were amplified from the products in the corresponding lanes of (a). Reprinted from Matsuda et al. (2005) with permission from Elsevier

Figure 8.4 Amplification products of the primary and nested PCR. Amplified products were electrophoresed on 3% agarose gel and stained with ethidium bromide. (a) Primary PCR with outer primers (L14734/H15863): M, HaeIII digest marker (TaKaRa); lane 1, amplified product from human DNA; lane 2, chimpanzee; lane 3, gorilla; lane 4, Japanese monkey; lane 5, crab-eating monkey; lane 6, pig; lane 7, cow; lane 8, dog; lane 9, goat; lane 10, chicken; lane 11, rat; lane 12, tuna; NC, negative control (no template DNA was added). (b) Nested PCR with inner primers (L15674/H15782): PCR products were amplified from the products in the corresponding lanes of (a). Reprinted from Matsuda et al. (2005) with permission from Elsevier

Coble, 2001). When these very rare types are observed in both evidence and reference samples, it increases the likelihood that the two samples are from the same source. The increased likelihood of a positive match may often be sufficient to make a definite identification, especially if taken in conjunction with additional physical and/or circumstantial evidence. However, there are some relatively common mtDNA types. In the same population, the frequency of the most common mtDNA type, '263G, 315.1C' is 7%, and there are 13 additional mtDNA types with frequencies of 0.5% or larger (Parsons and Coble, 2001). If both the evidence and reference samples contain the same relatively common type of mtDNA, it does not increase the likelihood of the two samples having come from the same source. Thus, we must seek further biological evidence that will allow us to make a more definite conclusion regarding whether the two samples match. While the probabilities at individual STRs can be multiplied to obtain the total likelihood of a match, the HV-I and HV-II loci must be treated as a single locus, thereby reducing the power of obtaining a highly significant match.

To increase individual discrimination power, polymorphisms residing outside two hypervariable regions (HV-I and HV-II) have been increasingly explored. One approach, which has been promoted by SWGAM (the Scientific Working Group on DNA Analysis Methods) (Allard et al., 2005) and by Carracedo and his colleagues (Quintans et al., 2004), is to expand targeting regions from the 600 bp HV-I and HV-II region to the entire 1100 bp control region. Using this approach, over 200 SNP sites were found, and, based upon the SNP profiles, phylogenically related mtDNA were grouped into haplo-groups. The term 'SNP' was initially introduced to indicate a single DNA base substitution that is observed with a frequency of at least 1% in a given population. Currently, however, this term refers to any SNP, including insertions and deletions, and the 1% frequency prerequisite has been eliminated. It is noted that, with the exception of length polymorphisms (C-stretch), HV-I and HV-II polymorphisms are regarded as a collection of densely clustered SNPs.

Another attempt to increase individual discrimination power is to profile SNPs across the entire mtDNA. Even though the density of SNPs in the coding region is low due to functional restraints, the coding region is 14 times larger than the control region, therefore the number of SNPs in the coding region is comparable to the number of SNPs found in the control region. However, it is impossible to sequence the entire mtDNA, therefore the accumulation of data regarding SNP positions over the entire mtDNA (Parsons and Coble, 2001; Hall et al., 2005) and the selection of an efficient method to spot the targeted SNPs (Sobrino et al., 2005) will increase individual discrimination power considerably. Among various methods applicable to such a task, the authors feel that microarray (Divne and Allen, 2005) is the most promising in that it allows fast simultaneous detection of a large number of SNPs.

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