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CRIME LABORATORY DIGF..5R Volume 20, Number 4 October 1993 If you have issues viewing or accessing this file contact us at NCJRS.gov. u.s. Department of Justice Federal Bureau of Investigation • • CRIME LABORATORY DIGF..5r Volume 20, Number 4 October 1993 • Guidelines for the Use of Mitochondrial DNA Sequencing in Forensic Science • Selected Topics in Forensic Voice Identification • CRIME LABORATORY DIGEST Volume 20, Number 4 October 1993 ARTICLES Guidelines for the Use of Mitochondrial DNA Sequencing in Forensic Science btJ Mark R. Wilson, Mark Stoneking, Mitchell M. HojIl91Jd, Joseph A. DiZinno, and Bruce Budowle It/to'17/ Selected Topics in Forensic Voice Identification by Bruce E. Koen~g 14 ~ Cj 9J- DEPARTMENTS • Editor's Note Book Review Employment Opportunity Meeting Announcements Special thanks to Glenda H. Wilson for providing her original artwork, which is depicted on the cover of this issue . The Crime 1.Jlboralory Digest (ISSN 0743-1872) is a quarterly forensic science journal published by the FBI laboratory Division in cooperation with the American Society of Crime laboratory Directors (ASCLD). It is intended to serve as a means of communication between crime laboratories, permitting • information of interest and value to be disseminated among crime laboratory scientists. Postmaster. Send address changes to Editor, Crime 1.Jlboralory Digest, FSRTC, FBI Academy, Quantico, VA 22135. • 146991- U.S. De,partment of Justice 146992 National Institute of Justice This document has been reproduced exactly as received from the person or organization originating it. Points of view or opinions stated in this document are those of the authors and do not necessarily represent the official position or policies of the National Institute of Justice. Permission to reproduce this I de ;J material has been granted by Public Domain/FBI u.s. Department of Justice • to the National Criminal Justice Reference Service (NCJRS). Further reproduction outside of the NCJRS system requires permission of the~ owner. • Guidelines for the Use of Mitochondrial DNA Sequencing in Forensic Science • Mark R. Wilson Mitchell M. Holland Federal Bureau of Investigation Armed Forces Institute of Pathology Washington, DC Washington, DC Mark Stoneking Joseph A. DiZinno Pennsylvania State University Federal Bureau of Investiga tion University Park, PA Washington, DC Bruce Budowle Federal Bureau of Investigation Quantico, VA Human mitochondrial DNA (mtDNA) is an extrachro­ assist in the differentiation of individuals in the human mosomal, closed circular, organelle-specific genome con­ population. It has been estimated that among Caucasians sisting of approximately 16.5 kb. The mtDNA genome has there is an average of1 nucleotide difference every 100 bases been completely sequenced (Andersoneta1.1981). Itconsists (1 %) in the most variable regions of mtDNA (Cann et al. of coding sequences for 2 ribosomal RNAs, 22 transfer 1987). This average is higher in individuals of African RNAs, 13 proteins, and a noncoding region apprOXimately descent, being approximately 2.3% (Vigilant et al. 1991). 1,100 base pairs long, called the dispJacementloop (D-Ioop) In the context of forensic science, the main advantage of or control region. Most of the sequence variation between typing mtDNA over nuclear DNA is its high copy number. individuals is found within the control region (Greenberg et While each set of nuclear chromosomes is present in only al. 1983). The double-stranded mtDNA molecule is com­ two copies per cell, mtDNA is present in hundreds or prised of one purine-rich strand and one pyrimidine-rich thousands of copies per cell. In cases where the amount of strand, designated the heavy chain and light chain, respec­ extracted DNA is very small or degraded, as in tissues such tively. Nucleotide positions in the mtDNA genome are as bone, teeth, and hair, the probability of obtaining a DNA • numbered according to the convention of Anderson et al. typing result from mtDNA is higher than that of polymor­ lJ 981). The origin of replication of the heavy strand begins phic markers found in nuclear DNA. the numerical designation of each base pair, continuing In addition to its higher copy number and resultant around the circle for approximately 16,569 base pairs. increased sensitivity, mtDNA is maternally inherited (Case An important feature of mtDNA, which simplifies DNA and Wallace 1981; Giles et al. 1980; HutchinsoI'. et al. 1974). sequencing, is its monoclonal nature. With the exception of Barring mutation, the mtDNA sequence is identical for certain disease conditions where tissue-specific deletions of siblings and all their maternal rela tives. This characteristic is large segments of the mitochondrial genome have been helpful in forensic cases where known maternall'ela tives can detected, for practical purposes all copies of an individual's provide reference samples for direct comparison to the mtDNA sequence are identical (Monnat and Loeb 1985; questioned mtDNA type (GinUler et al. 1992; Holland et fil. Monnat et al. 1985; Monnat and Reay 1986). Nuclear DNA, 1993; Stoneking et al. 1991). however, is diploid, consisting of a maternally inherited set An intriguing aspect of mtDNA which has recently been of chromosomes and a paternally inherited set of chromo­ cited in the literature as a possibility is predicting the ethnic I somes. In order to unambiguously sequence nuclear DNA background of a DNA donor by his or her mtDNA sequence (with the exception of the monoclonal Y chromosome), the (Horaietal.1993). It appears thatspecificmtDNA sequences two alleles must first be separated. are found in some ethnic groups but not in others. It may be Due to its urrique mode of inheritance, mtDNA does not possible to determine the ethnic bilckground of an indi­ undergo recombination. However, the low fidelity of m tDNA vidual by his or her mtDNA sequence. Appropriately polymerase and the apparent lack of mtDNA repair mecha­ defined sta tistical estimates should always accompany such nisms have led to a higher rate of mutation in the mitochon­ assessment of ethnicity. drial genome. Some regions of the mtDNA genome appear Because of the potential for single nucleotide substitu­ to be evolving at5 to 10 times the rate of single copy nuclear tions in mtDNA, a single base could represent the difference genes (Brown et al. 1982; Cann et al. 1987). These areas of between a match and an exclusion in a forensic case. There­ relatively high mutability are still well conserved. It is these fore, careful gUidelines for determining the means by which regions which are of forensic interest, due to their ability to sequences are validated and reported must be established. • 68 Crime Laboratory Digest ------------.~------------------------------------------- Mitochondrial DNA typing, using a variety of methods, has pooled togeUler (in the case of dye-labeled primers). In proven to be valuable as a means for human identification in either case, the sequencing products are loaded on a many forensic cases (Higuchiet al. 1988; Hopgoodet al. 1992; sequencing gel in a single lane. Automated sequencing with Orrego and King 1990; Stoneking et al. 1991). In some of fluorescent labels allows co-electrophoresis of each of the • these cases, mtDNA specific oligonucleotide hybridization four termination reactions, thereby reducing the number of assays have been employed (Stoneking et af. 1991); manual lanes which must be run per template from four to one. sequencing has been used in others (Ginther et al. 1992; Cycle sequencing is a technique which combines the Higuchi et al. 1988; Holland et al. 1993; Orrego and King dideoxy terminator chemistry with the sensitivity of PCR 1990), and another group has reported research into fluores­ (Carothers etal.1989; Murray 1989). The DNA templates are cence-based automa ted sequencing of m tDNA (Hopgood et cyclic1y denatured and annealed with sequencing primers al. 1992; Sullivan et al. 1992). In order to establish mtDNA which are extended in the presence of a thermally stable typing as a method for large scale, routine forensic applica­ enzyme, such as Taq DNA polymerase. The reaction con­ tions, it is proposed that some basic guidelines be adopted. tains labeled primers, terminators, or a dNTP. Each frag­ ment is uniquely labeled through the use of ddNTPs, as discussed previously. With each cycle the labeled fragmen ts are denatured and removed from the template, which is DNA Sequencing again available for primer annealing, extension, and termi­ nation. Each template molecule is available for repeated use Two distinct methods for sequencing DNA were pub­ to generate a new sequencing product with each cycle. The lished in the late 1970's (Maxam and Gilbert 1977; Sanger et sequencing productis thereby amplified, resulting in greater al.1977). The chain termination method, or Sanger method, sensitivity with a limited amount of the DNA template. is the more widely used. The Sanger method utilizes an Because the DNA template is thermally denatured prior to oligonucleotide primer which is annealed to the DNA tem­ primer annealing, double-stranded DNA can be used as a plate to be sequenced. The primer is extended by a DNA template in cycle sequencing reactions. This advantage polymerase in the presence of buffer, salts, deoxynucleotides obviates the need to generate single-stranded DNA from (dNTPs), and dideoxynucleotides (ddNTPs). When a ddNTP amplified PCR products prior to sequencing, thereby simpli­ is incorpora ted into the growing DNA strand, the chain can fying sample preparation. Cycle sequencing can be em­ no longer be
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