ABSTRACT
In the first ever systematic genetic survey, we have used rigorous decontamination followed by mitochondrial 12S RNA sequencing to identify the species origin of 30 hair samples attributed to anomalous primates. Two Himalayan samples, one from Ladakh, India, the other from Bhutan, had their closest genetic affinity with a Palaeolithic polar bear, Ursus maritimus. Otherwise the hairs were from a range of known extant mammals.
Subject(s)
Hair/physiology , Primates/genetics , RNA, Ribosomal , Animals , Bhutan , Fossils , Genetic Testing , India , Molecular Sequence Data , RNA , RNA, Mitochondrial , Ursidae/geneticsABSTRACT
BACKGROUND: Mitochondrial DNA analysis is typically applied to degraded skeletal remains and telogen or rootless hairs. Data on the application of the method to very small hairs less than 0.5 cm from an age-matched and -challenged sample set are lacking. METHODS: One hundred fourteen hairs sized less than 1 cm from a 1993 case were analyzed for mitochondrial DNA according to laboratory standard operating procedures. For some hairs, a screening approach was applied, which permitted some samples, such as victim hairs on victim clothing, to be eliminated from the process quickly. Degraded samples were amplified with "mini-primers," and 12S species testing was applied when non-human hairs were encountered. RESULTS: Partial to full control region human mitochondrial DNA profiles or species identifications (non-human species) were obtained from 93% of hairs under 1 cm, 92% of hairs under 5 mm, and 90% of hairs under 3.5 mm. Nineteen of 21 hairs 2 mm or less gave full or partial profiles. Among 128 hairs of all sizes tested in the case, 9 gave no results, 3 were canine in origin, and 73 did not exclude six known individuals tested in the case. Twenty-two hairs had nine additional profiles that were observed two or more times each. Twenty-one hairs showed singleton types not matching each other or any individual. CONCLUSIONS: Crime scene hairs that are both aged and small are often judged to be unsuitable for either hair microscopy or DNA analysis. This study of age-matched challenged small hairs indicates that even the smallest probative crime scene hairs are suitable for mitochondrial DNA analysis and can provide useful data.
ABSTRACT
Since July 2004, Mitotyping Technologies has been amplifying and sequencing a approximately 150 base pair fragment of mitochondrial DNA (mtDNA) that codes for 12S ribosomal RNA, to identify the species origin of nonhuman casework samples. The approximately 100 base pair sequence product is searched at http://www.ncbi.nlm.nih.gov/BLAST and the species match is reported. The use of this assay has halved the number of samples for which no mtDNA results are obtained and is especially useful on hairs and degraded samples. The availability of species determination may aid forensic investigators in opening or closing off lines of inquiry where a highly probative but challenging sample has been collected.
Subject(s)
DNA Fingerprinting/methods , DNA, Mitochondrial/genetics , RNA, Ribosomal/genetics , Species Specificity , Animals , DNA Primers , Deer/genetics , Dipodomys/genetics , Dogs/genetics , Humans , Mice/genetics , Polymerase Chain Reaction , Puma/genetics , Sciuridae/genetics , Sequence Analysis, DNAABSTRACT
Between February 1999 and May 2005, 116 DNA extractions were completed on skeletal remains from routine casework. Overall, at least a partial mitochondrial DNA (mtDNA) profile was obtained on 83.6% of samples. Skeletal remains fell into two general categories: (1) samples for body identifications submitted by law enforcement and (2) samples submitted to answer historical or family identity questions. Body identification cases were more likely to yield full mtDNA profiles, whereas historical cases were more likely to result in partial profiles. Overall, the ability to obtain a full or partial profile primarily reflects the difference in the average age and condition of the samples in these two categories and thus, difference in the quantity and quality of the DNA. Cremated remains were uniformly unsuccessful, whereas infant/fetal remains were uniformly successful. Heteroplasmy in skeletal remains was observed at a rate similar to that in hair ( approximately 10%). For body identification cases, skeletal remains had the same mtDNA profile as the accompanying reference sample in 50% of cases.
Subject(s)
DNA, Mitochondrial/isolation & purification , Forensic Anthropology , Adolescent , Adult , Aged , Aged, 80 and over , Bone and Bones/pathology , Burns/pathology , Child , Child, Preschool , Complementarity Determining Regions/genetics , DNA Degradation, Necrotic , DNA Primers , Humans , Infant , Middle Aged , Polymerase Chain Reaction , Sequence Analysis, DNA , Tooth/pathologyABSTRACT
Mitochondrial DNA (mtDNA) sequencing can provide crucial information to forensic investigators when the quantity and quality of DNA would otherwise be limiting. The difficulty of analyzing mtDNA mixtures, however, has been a significant obstacle to its broader use in forensics. Denaturing high-performance liquid chromatography (DHPLC) in combination with direct sequencing makes it possible to determine the linkage phase of individual amplicons in a mixture. The reliability of the approach is based, in part, on the strong correlation between a change in the relative quantities of different DNA amplicons in a given mixture versus a change in the relative electrophoretic peak heights at mixed base positions. Using standard operating procedures previously validated for use in forensic laboratories, this approach enables sequence-specific fractionation of natural (heteroplasmic) or situational (multi-contributor) DNA mixtures prior to direct sequencing. As a novel approach for the rapid and accurate analysis of DNA mixtures, DHPLC may aid criminal investigators by making it possible to obtain definitive mitochondrial DNA results from otherwise challenging samples.
Subject(s)
Chromatography, High Pressure Liquid/methods , DNA, Mitochondrial/genetics , DNA, Mitochondrial/isolation & purification , Forensic Genetics/methods , Electrophoresis , Humans , Nucleic Acid Denaturation , Reproducibility of Results , Sequence Analysis, DNA/methodsABSTRACT
A five year retrospective review of mitochondrial DNA (mtDNA) analysis on 691 casework hairs was carried out. A full or partial mtDNA profile was obtained for > 92% of hairs. With increasing age of the hair, the likelihood of obtaining a full profile decreased, although "mini-primer sets" could often be used to capture a partial profile. With increasing color and diameter of the hair, the likelihood of obtaining a profile increased. Full or partial profiles were obtained on more than 80% of 114 hairs < or = 1.0 cm. Mixtures were observed in 8.7% of hairs tested; mixtures increased with the age of the hair and were presumed to be due to exterior surface contamination that could not be sufficiently cleaned prior to extraction, since the overall level of laboratory contamination was low. The frequency of sequence heteroplasmy was 11.4%, and both hot-spot and novel sites were observed. In about one-third of these observations, another sample in the case showed either the same heteroplasmic site or a nucleotide substitution at that site.
