Southwestern Institute of Forensic Sciences Criminal Investigation Laboratory

Forensic Biology Unit Mitochondrial DNA Training Program, Version 1.0

Authorizations: Date: Timothy J. Sliter, Ph.D., Senior Forensic Biologist Date: Robert A. Poole, Chief of Physical Evidence Date: Jim Dempsey, Quality Manager

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Overview This protocol describes the training program that will be undertaken by analysts in order to be qualified to perform DNA profiling of mitochondrial DNA (mtDNA) using the methods described in the procedure manual Mitochondrial DNA Sequence Analysis Protocols. The training program consists of the following components, some of which may be done concurrently: 1. 2. 3. 4. 5. 6. Readings on the theoretical and technical basis of mtDNA profiling, and its forensic applications. A combination of formal and informal instruction from a qualified analyst on technical and theoretical issues in mtDNA profiling. Technical instruction by a qualified analyst in the procedures of mtDNA profiling. Technical exercises on practice samples representing a range of samples typical of casework material. A knowledge-based competency test. A technical competency test.

Elements of the training may be modified or eliminated based upon the previous training and experience of the trainee. The trainee will maintain a comprehensive notebook of training materials and results, and will meet periodically with a trainer to assess progress toward training objectives. A record of progress through the training will be maintained in the form of a training checklist that will be completed by the trainer.

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Readings The trainee will read and discuss with the trainer the following materials, which are maintained in a training notebook: Anderson, S. et al., Sequence and organization of the human mitochondrial genome. Nature 290: 459-465 (1981). Bendall, K. and Sykes, B.C., Length heteroplasmy in the first hypervariable segment of the human mtDNA control region. American Journal of Human Genetics 57: 248-256 (1995). Bendall, K. et al., Heteroplasmic point mutations in the human mtDNA control region. American Journal of Human Genetics 59: 1276-1287. Bendall, K. et al., Variable levels of a heteroplasmic point mutation in individual hair roots. American Journal of Human Genetics 61: 1303-1308 (1997). Federal Bureau of Investigation. MtDNA Interpretation (unpublished). Gill, P. et al., Identification of the remains of the Romanov family by DNA analysis. Nature Genetics 6: 130-135 (1994). Ginther, C. et al., Identifying individuals by sequencing mitochondrial DNA from teeth. Nature Genetics 2: 135-138. Gock, C.D. et al., Transmission of mitochondrial DNA heteroplasy in normal pedigrees. Human Genetics 102: 182-186 (1998). Holland, M.M. et al., Mitochondrial DNA sequence analysis of human skeletal remains: Identification of remains from the Vietnam war. Journal of Forensic Sciences 38:542-553 (1993). Holland, M.M. and Tully, L.A., Mitochondrial DNA (preprint) (1999). Holland, M.M. MtDNA match criteria. In Mitochondrial DNA Sequence Analysis in Forensic Casework: Methods and Current Issues (preprint) (1998). Ivanov, P.L. et al., Mitochondrial DNA sequence heteroplasmy in the grand duke of Russia Georgji Romanov establishes the authenticity of the remains of Tsar Nicholas II. Nature Genetics 12: 417-420 (1996). Jazin, E. et al, Mitochondrial mutation rate revisited: Hot spots and polymorphisms. Nature
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Genetics 18: 109-110 Jeneuth, J. P. et al, A random genetic drift in the female germline explains the rapid segregation in mammalian mitochondrial DNA. Nature Genetics 14: 146-151 (1996). Johns, D.R. Mitochondrial DNA and disease. New England Journal of Medicine 333:638-644 (1995). Kadenbach, B. et al., Human aging is associated with stochastic somatic mutations of mitochondrial DNA. Mutation Research 338: 161-172 (1995). Manfredi, G. et al., the fate of human sperm-derived mtDNA in somatic cells. American Journal of Human Genetics 61: 953-957. Marchington, D.R. et al., Homopolymeric tract heteroplasmy in mtDNA from tissues and single oocytes: Support for a genetic bottleneck. American Journal of Human Genetics 60: 408-416 (1997). Michaels, G.S. et al., Mitochondrial DNA copy number in bovine oocytes and somatic cells. Developmental Biology 94: 246-251 (1982). Ozawa, T. Mechanism of somatic mitochondrial DNA mutations associated with age and diseases. Biochimica et Biophysica Acta 1271: 177-189. Parson, T.J. et al., A high observed substitution rate in the human mitochondrial DNA control region. Nature Genetics 15: 363-368 (1997). Parsons, T.J. and Holland M.M., Mitochondrial mutation rate revisited: Hot spots and polymorphisms - Reply. Nature Genetics 18:109-110 (1998). Parsons, T.J., MtDNA biology and the Interpretation of mtDNA sequence evidence. In Mitochondrial DNA Sequence Analysis in Forensic Casework: Methods and Current Issues (preprint) (1998). Wilson, M. et al., Extraction, PCR amplification and sequencing of mitochondrial DNA from human hair shafts. Biotechniques 18: 662-669 (1995). Wilson, M.R. et al., Guidelines for the use of mitochondrial DNA sequencing in forensic science, Crime Laboratory Digest 20: 68-77 (1993). Wilson, M. et al., A family exhibiting heteroplasmy in the human mitochondrial DNA control region reveals both somatic mosaicism and pronounced segregation of mitotypes. Human
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Genetics 100: 167-171 (1997).

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Instruction Instruction will be provided to the trainee on the topics listed below. Instruction will be provided by a trained analyst, and will consist of either formal lectures, informal instruction, or a combination of the two. Elements of the instruction may be modified or omitted based upon the previous education and experience of the trainee. Alternatively, this element of the training program may be satisfied by the trainee taking a workshop or course in mtDNA profiling that is approved by the laboratory management for this purpose. 1. 2. 3. 4. 5. 6. mtDNA molecular biology and genetics Extraction methods for mtDNA Polymerase chain reaction mtDNA amplification DNA sequencing mtDNA sequence interpretation issues a. Homopolymeric tracts b. Heteroplasmy c. Defining concordance d. Source attribution mtDNA database and statistical issues a. Counting method b. Confidence limits QC/QA issues in mtDNA profiling Report writing

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8. 9.

Technical Instruction The trainee will receive technical instruction in the following laboratory procedures from a trainer: 1. 2. 3. 4. 5. 6. 7. 8. DNA extraction methods Quantitation of DNA with the QuantiBlot kit Amplification of mtDNA Product gel analysis of amplified mtDNA Sequencing of mtDNA templates ABI 310 Genetic Analyzer operation Interpretation of mtDNA sequence data Performing database searches & statistical analysis

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Technical exercises The trainee will conduct mtDNA profiling of a minimum of 25 practice samples. Profiling will consist of analysis of both HV1 and HV2. The samples will consist of hair and standards from known individuals (laboratory personnel, family members, etc). The samples tested are to include a variety of types of hairs, such as chemically treated hair, fine hair, infant hair, hair with dried blood from a different source, and hair mounted on a microscope slide. The samples should also include at least one set of samples from multiple generations of the same family. Knowledge-base qualifying test The trainee will be administered a knowledge-based qualifying test. The test will be reviewed by the trainer, and scored as either satisfactory or unsatisfactory. In the case of an unsatisfactory result, the trainee will be provided an opportunity to retake the test after additional instruction/preparation time. Technical qualifying test The trainee will be administered a technical qualifying test consisting of either samples from an old external proficiency test, or internally generated samples. The trainee will perform mtDNA profiling on the samples, perform statistical analyses, and generate a report of findings. The results will be reviewed by the supervisor, and judged to be either satisfactory or unsatisfactory. In the event that the results are unsatisfactory, the trainee will be provided additional time for practice samples, and the test will then be administer again. External proficiency test Successful completion of the qualifying tests will qualify the analyst to perform independent case work. Within 6 months of qualification the analyst must complete an external proficiency test. Mock Trial (optional) Depending upon prior testimony experience, the analyst may be provided the opportunity to participate in a mock trial prior to his/her first official testimony. In the case of in-house mock trials, feed back will be given by the supervisor, section chief, and DNA analytical staff.

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mtDNA Training Checklist Trainee __________________________ Trainer __________________________ Date completed Trainer initials

Training component 1. Readings 2. Instruction 3. Technical Instruction 4. Practice exercises 5. Knowledge-based competency test 6. Technical competency test 7. External proficiency test

Notes

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