Mitochondrial DNA amplification success rate as a function of hair morphology

This study examines the amplification success rate of mitochondrial DNA from human head hair with respect to their potential for forensic application. Mitochondrial DNA was isolated using a Chelex-based extraction method and amplified using the LINEAR ARRAY duplex PCR system. The particular focus of this study was to characterize the morphological features of human head hair in order to further the understanding of the factors that influence amplification success rate in hair tissue using the LINEAR ARRAY duplex PCR system. 2554 head hairs from 132 individuals representing four population groups were amplified. The hair samples were characterized as follows: 1251 were identified microscopically as telogen hairs and 1303 were classified as hairs without roots (removed before extraction). Amplification success was assessed as a function of several independent variables: morphological characteristics; telogen root versus no root; donor age; scalp origin; use of cosmetic hair treatments; and race of the donor. The results show that a positive correlation exists between amplification success and the presence of a telogen root. Combining the amplification success with either the original or optimized protocol, telogen hairs result in an overall success rate of 77.5% compared with 65% for hairs with no roots. Controlling for telogen hairs, the findings indicate that the overall success rate is independent of cosmetic hair treatments; medulla structure; shaft length, diameter, and volume; and scalp origin. Conversely, the age of the donor, the race of the donor, and hair pigmentation all contribute to a variation in amplification success rate.     

Characterization of Mitochondrial DNA Sequence Heteroplasmy in Blood Tissue and Hair as a Function of Hair Morphology*,†,‡

Abstract: This study characterizes mitochondrial DNA (mtDNA) sequence heteroplasmy in blood tissue and hair as a function of hair morphology. Bloodstains (127 individuals) and head hairs (128 individuals) were typed using the mtDNA LINEAR ARRAY? assay. A total of 1589 hairs were interpreted: 1478 (93%) were homoplasmic and 111 (7%) exhibited heteroplasmy at one or more positions. Seventy‐one percent (82/116) of individuals were homoplasmic, whereas 29% (34/116) exhibited heteroplasmy in at least one hair. The results demonstrate intra‐ and inter‐tissue differences in heteroplasmy within individuals. Sequence heteroplasmy among hairs from each individual varied from 0 to 90%; the frequency does not differ significantly with population group, cosmetic treatment, age, gender, medulla morphology, region of the scalp, hair growth phase, or, when comparing living and deceased donors. However, the results support a correlation between heteroplasmy and hair pigmentation; typically, lighter‐pigmented hairs exhibit a higher incidence of sequence heteroplasmy compared to darker hairs.     

Mitochondrial DNA typing screens with control region and coding region SNPs

Mitochondrial DNA (mtDNA) analysis has found an important niche in forensic DNA typing. It is used with highly degraded samples or low-copy number materials such as might be found from shed hair or bones exposed to severe environmental conditions. The primary advantage of mtDNA is that it is present in high copy number within cells and therefore more likely to be recovered from highly degraded specimens. A major disadvantage to traditional forensic mtDNA analysis is that it is time-consuming and labor-intensive to generate and review the 610 nucleotides of sequence information commonly targeted in hypervariable regions I and II (HVI and HVII) of the control region. In addition, common haplotypes exist in HVI/HVII mtDNA sequences that can reduce the ability to differentiate two unrelated samples. In this report we describe the utility of two newly available screening assays for rapid exclusion of non-matching samples. The LINEAR ARRAY mtDNA HVI/HVII Region-Sequencing Typing Kit (Roche Applied Science, Indianapolis, IN) was used to type 666 individuals from U.S. Caucasian, African American, and Hispanic groups. Processing of the LINEAR ARRAY probe panels "mito strips" was automated on a ProfiBlot workstation. Observable variation in 666 individuals is reported and frequencies of the mitotypes within and between populations are presented. Samples exhibiting the most common Caucasian mitotype were subdivided with a multiplexed amplification and detection assay using eleven single nucleotide polymorphisms in the mitochondrial genome. These types of screening assays should enable more rapid evaluation of forensic casework samples such that only samples not excluded would be subjected to further characterization through full HVI/HVII mtDNA sequence analysis.     

Forensic casework analysis using the HVI/HVII mtDNA linear array assay

The mitochondrial hypervariable regions I and II have proven to be a useful target for analysis of forensic materials, in which the amount of DNA is limited or highly degraded. Conventional mitochondrial DNA (mtDNA) sequencing can be time-consuming and expensive, limitations that can be minimized using a faster and less expensive typing assay. We have evaluated the exclusion capacity of the linear array mtDNA HVI/HVII region-sequence typing assay (Roche Applied Science) in 16 forensic cases comprising 90 samples. Using the HVI/HVII mtDNA linear array, 56% of the samples were excluded and thus less than half of the samples require further sequencing due to a match or inconclusive results. Of all the samples that were excluded by sequence analysis, 79% could be excluded using the HVI/HVII linear array alone. Using the HVI/HVII mtDNA linear array assay, we demonstrate the potential to decrease sequencing efforts substantially and thereby reduce the cost and the turn-around time in casework analysis.     

Optimization of a duplex amplification and sequencing strategy for the HVI/HVII regions of human mitochondrial DNA for forensic casework

A duplex primer set for the amplification of mitochondrial DNA HVI and HVII control regions was evaluated for the optimization of a DNA sequencing protocol suitable for forensic casework. HVI and HVII products, with the absence of non-specific products, could be detected by agarose gel electrophoresis when as little as 0.5 and 0.1pg of DNA were amplified for 34 and 38 cycles, respectively. Because HVI and HVII amplicons are co-synthesized in the duplex PCR, fewer steps are required (lessening the risk of cross contamination events) and more frugal use of precious extracted DNA samples is possible, both desirable features for forensic casework. The ABI Prism BigDyetrade mark version 1.1 chemistry provided high quality sequencing data, with little or no background noise and uniform peak heights, outcomes that favored reliable detection of heteroplasmy, particularly at early sequence reads (<40 bases). Optimal compromise between sensitivity and sequence accuracy in the absence of noise was achieved starting at 150 mitochondrial genome copies. The protocol is effective (no sequence errors) with highly degraded DNA (average detectable template size of 200bp). Dual artificial template mixtures with the minor component at 15% suggests that heteroplasmy should be detected at this level with confidence.