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• • 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

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• 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 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 -rich strand and one -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 extended. Fragments of different lengths are ployed in both manual and automated formats. thereby generated, each fragment's size depending on the Fluorescence-based automated sequencers have high exact site in which chain elongation is terminated. Generally throughput and permit rapid analysis of sequence informa­ one of the dNTPs is radiolabeled, and hence all sequencing tion due to automa ted base calling. Through accompanying fragments are internally labeled prior to separation in dena­ editing software, these instruments allow the user to rapidly • turing polyacrylamide gels. The result is a ladder of frag­ compare sequences with one another, highlight the differ­ ments, differing by a minimum of one nucleotide, which ences among multiple sequences, and compare individual after autoradiography can be read as a sequence when all sequences with a reference standard. For these reasons fuis four termina tion reactions are run side-by-side. This type of approach is attractive to forensic science (Hopgood et al. sequencing, now called manual sequencing as opposed to 1992). However, caution must be exercised in using such automated sequencing, is comparatively more time con­ instruments for reasons to be discussed later. One current suming and labor intensive. manufacturer of automated DNA sequencers is Applied Polymerase chain reaction (PCR) amplified DNA tem­ Biosystems, Inc. (Foster City, CAl, which produces the plates can be sequenced either manually or through the use automated DNA sequencer 373A. (Applied Biosystems, Inc. of an automated sequencer. Although manual sequencing is is a Division of Perkin Elmer, Norwalk, CT). The present currently more widespread, automated sequencers provide discussion will be confined to the use of this instrument. high throughput and ease of data management through Two specific types of chemistries are employed with accompanying computer software. A number of different fluorescence-based sequencing: primer chemistry and ter­ sequencing strategies have been employed for sequencing minator chemil'wy. With primer chemistry, the fluol'e,<;cent PCR-generated templates (Ellingboe and Gyllensten 1992; label is attached to the 5' end of the sequencing primer. Four Gyllensten and Erlich 1988). Each method relies on the distinct labels are used, one for each dideoxy terminator Sanger did~xy terminator chemistry. . reaction. For instance, a blue fluorescently tagged primer Fluorescent automated DNA sequencing was intro­ might be used with the dideoxy terminator, green duced in 1986 (Smith et al. 1986). In these sequencing might be used with an adenosine terminator, etc. Four r.eactions, fluorescently labeled oligonucleotide primers or separate terminator reactions are then performed on the dideoxy nucleotide terminators are incorporated into a DNA DNA template to be sequenced, and the resultant products fragment which is then electrophoretically separated and are pooled prior to electrophoresis. Terminator chemistry detected by laser-induced fluorescence. The temporal, se­ places the fluorescent label on the dideoxy terminator itself quential passage of these fragments past the detector win­ so that each of the four termination reactions can occur dow gives rise to the sequence of the template. Since each simultaneously in the same reaction vessel. FollOWing a terminator or primer can be uniquely labeled, all four termi­ purification step to remove unincorporated dideoxy termi­ nation reactions can be performed together (in the case of na tors, the products are directly loaded OntO the sequencing • dye-labeled terminators) or performed separately and then gel containP£i within the instrument. Volume 20 Number 4 October 1993 69 As electrophoretic separation of labeled fragments is strand sequence, as opposed to the complementary heavy progressing, real time detection, in the form of electronic strand of the double-stranded native DNA. When a differ~ signals, is sent to a computer to be analyzed. A laser scans ence between an individual's sequence and the Anderson the polyacrylamide gel plate once every 4 seconds. The sequence is observed, the difference is sometimes referred to electronic signals result from laser excitation and fluores­ asa "polymorphism with respect to the Andersonsequence." • cence of the separated fragments as they pass by a detector For example, position number 16189 in the Anderson se­ window. A photomultiplier tube within the instrument quence lists the nucleotide thymidine or T. However, in converts the optical signal into an electronic signal. A typical some other individuals sequenced, that particular position electrophoretic run lasts approximately 9 hours. Therefore, contains a cytosine or C. If all other nucleotide positions a tremendous amount of information, comprising about 12 were identical to the Anderson sequence, this individual's megabytes, is generated from each sequencing run. This mtDNA type would be referred to as 16189-C, and it could body of information is termed the gel file. It reprC3ents data be assumed that all other positions in the region sequenced points from across the gel collected at 4-second intervals other than 16189 are identical to the Anderson sequence. over the duration of the run. The computer begins its Defined starting and stopping positions for mtDNA analysis of the data from the information cont

I!

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) V\.

Figure 1. A limited portion of a mtDNA sequence obtained from HV1 of the control region of mtDNA through the cycle sequencing technique. The peaks represent the sequential passage by the detector window of mtDNA fragments that differ in length by one base. Fluorescence detection of the terminafor dye on each fragment permits a detemlination of the final base incorporated into each fragment. The order of bases in this short sequence is shown at tire top of tIre chromatogram. TIre result is the sequence of tire template DNA, which in tltis case was a peR product amplified from a human hair shaft extract.

thymidine and five additional cytosines. In some individu­ Confirmation of DNA Sequences als, an extra cytosine is observed in the initial stretch of seven cytosines, resulting in eight cytosines prior to the sole thymi­ Each sequence obtained should be confirmed, dine. The insertion could, by convention, be recorded after whether for databasing purposes or forensic casework. For the final cytosine in the initial run of seven and hence be example, if the light strand is sequenced and results in designa ted as 309.1 C. Should the run of cytosines be longer sequence A, the heavy strand can be sequenced and would than eight (for example, nine), prior to the thymidine, the result in sequence B. When the reverse complement of type would be recorded as 309.1 C, 309.2 C. Deletions would sequence B is compared with sequence A, the two should be simply be recorded as the number of the base or bases identical. Alternatively, the same strand can be sequenced missing with respect to the Anderson nomenclature (for with the same or a different sequencing primer, indepen­ example, D315 or D315,316). Experience will determine dently confirming the initial sequence inform a tion from the whether this shorthand method of listing mtDNA types will same strand. Should any ambiguities arise between the prove to be a more effective manner of storing mtDNA original sequence and the confirmatory sequence, the DNA • sequence information. strand could be sequenced repeatedly until the ambiguity is Volume 20 Number 4 October 1993 71 resolved (or the ambiguity could be designated as an N, Validation meaning that the ambiguity is retained). Figure 2 shows a completed database sequence compiled using the Applied A number of specific considerations should be ad­ Biosystems, Inc. software package entitled SeqEd. Both dressed before mtDNA sequencing is used in forensic case­ strands of the template are sequenced, and the reverse work. They are as follows: • complement of the heavy strand sequence is directly com­ pared to the light strand sequence for confirmation. 1. If database sharing among laboratories is to be accom­ The ultimate goal of forensic DNA sequencing should p~ished, each sequencing stra tegy should yield identical be to optimize each set of sequencing reactions so that no results for the same DNA sequencing templates. For sequence ambiguities remain. However, when sequencing example, laboratories that sequence using manual tech­ samples from forensic casework, complete, unambiguous niques should share DNA templates with laboratories sequencing results may not always be possible, even after that utilize automated sequencers. Since automated confirmation. For example, PCR amplification of mtDNA sequencing can be done with dye terminators or dye extracted from liquid blood is generally robust and can result primers, each of these methods should be compared in Iargesegrnents of amplified product. These large amplicons and demonstrated to yield similar sequencing results on can be sequenced repeatedly using different sequencing shared DNA templates within laboratories and between primers or types of chemistries. As a result, one large laboratories. amplicon could yield enough DNA template for repeated 2. Maternal inheritance of mtDNA must be experimen­ sequencing of different regions or the same region within the tally verified if it is to be used as a means of establishing amplicon. Conversely, forensic samples such as bone and identity in forensics. As maternal inheritance has been hair may not yield enough mtDNA to generate amplification well documented, this verification should be minimal, of such large peR products. However, these samples might encompassing a small number of families and showing yield short PCR fragments of approximately 200 to 300 base that sequence information is identical from maternally pairs. Sequencing these short amplicons may require alter­ related individuals. na te conditions, as the size of the DNA templa te is a variable 3. A number of tissue types from the same individual to be considered in the process of optimizing a sequencing should be used for DNA extraction and sequencing to reaction. show that an individual's mtDNA sequence is identical Although it would be desirable to sequence an entire from tissue to tissue, regardless of the somatic origin of amplicon without any ambiguities, this may not be practi­ the extracted mtDNA. Studies with human hairs should cable. However, this is not necessary in order to acquire show tha t different hairs from the same individual yield some informa tion of forensic utility. One way tha t sequence identical mtDNA sequences. ambiguities may be treated when compared to a mtDNA 4. Environmental insult studies, such as exposure of database will be discussed later. samples to sunlight, soil, etc., should be performed to •

SF 7026 HV2* 11° 21~ 1° )0 51° ,__ 6l_._. ___ ,_ ... ~L. ___ ., __ ._~ 1 SF 7026-C GTGCACGCGA TAGCATTGCG AGACGCTGGA GCCGGAGCAC CCTATGTCGC AGTATCTGTC TTTGATTCCT GCCCCATCCC ~ SF 7026-11 GTGCACGCGA TAGCATTGCG AGACGCTGGA GCCGGAGCAC CCTATGTCGC AGTATCTGTC T'l'TGATTCCT GCCCCATCCC 3 *------~------*-----* 4 ANDERSON ATGCACGCGA TAGCATTGCG AGACGCTGGA GCCGGAGCA~ CCTATGTCGC AGTATCTGTC TTTGATTCCT GCCTCATCCT 9fl,~0:lO .:.e___ .. _ 1e. ___. , _ 11° 110 11° ATTATTTATC GCACCTACGT TCAATATTAC AGGCGAACAT ACTTACCAAA GTGTGTTAAT TAATTAATGC TTGTAGGACA ATTATTTATC GCACCTACGT TCAATATTAC AGGCGAACAT ACTTACCAAA GTGTGTTAAT TAATTAATGC TTGTAOGACA

ANDERSON ATTATTTATC------GCACCTACGT------TCAATATTAC AGGCGAACAT------ACTTACTAAA------*--- GTGTGTTAAT------TAATTAATGC ------TTGTAGGACA 11° 11° 11° ~~o , 2}0 , .. T,_,_.~ __ ,"~l~ ____.T_ 1 SF 7026-C TAATAATAAC AATTGAATGT CTGCACAGCC GCTTTCCACA CAGACATCAT AACAAAAAAT TTCCACC&~ CCCCCCCTCC 2~SF 7026-0 TAATAATAAC AATTGAATGT CTGCACAGCC GCTTTCCACA CAGACATCAT AACAAAAAAT TTCCACCAAA CCCCCCCTCC 3 *------f ANDERSON TAATAATAAC AATTGAATGT CTGCACAGCC ACTTTCCACA CAGACATCAT AACAAAAAAT TTCCACCAAA CCCCCCCTCC 2t O ,21 0 21° 3t: /1 BF 7026-C CCCCGCTTCT GGCCACAGCA CTTAAACAC ~ BF 7026-0 CCCCGCTTCT GGCCACAGCA CTTAAACAC ---*------~ ANDERSON CCC-GCTTCT GGCCACAGCA CTTAAACAC

Figure 2. Confirmatory sequence infom/ation can be generated by directly comparing the light strand (7026-C) ofllUman mtDNA witll • tile reverse complement of the heavy strand (7026-D) of the same region sequenced with a separate primer. Automatic reverse complementary alignment simplifies handling of sequence data and allows rapid confirmation of template sequence. Differences from tlte reference (Anderson) sequence are highlighted by an asterisk.

72 Crime Laboratory Digest show that mtDNA sequence data is preserved and can Population Frequency Calculations be reliably typed. Studies on human hairs, for example, should include hairs exposed to dyes, bleaches, and Once a mtDNA type has been established, a determina­ other common hair treatments. As the chemistry of tion of the frequency of this type in the general population DNA is very well understood, it should be noted that if could be presented in order for a jury to assess the weight of • an extracted DNA sample enzymatically amplifies and the evidence in a courtroom trial. General databases, which sequences, if properly analyzed the sequence informa­ take into considera Hon the inherent genetic dissimilarities of tion correctly reflects that of the original extract. the major races of man, should be compiled. These databases 5. With fluorescence-based automated DNA sequencers, a should include samples collected from the general popula­ known positive control should be sequenced with each tion and generally should be divided into Caucasian, Afri­ sequencing run. This yields information on the general can, and Asian racial groups. An example of one form of quality of the sequencing reagents, the efficiency of such a database is shown in Figure 3 (Stoneking et al. 1991). unincorporated label removal, and the strength of the As the mitochondrial genome is inherited in its entirety fluorescent signal. This lane thereby represents quality without recombination, a complete mtDNA sequence is the control for each sequencing run. This positive control equivalent of a single genetic locus. Therefore, the frequency should be of the same general physical form as the DNA of a mtDNA type might be multipHed by allele frequencies sequenced from the unknown sample. This means that of polymorphic nuclear loci, but at this time mtDNA se­ if double-stranded template DNA is used in a cycle quence information from within an individual cannot be sequencing format, a double-stranded sequencing con­ multiplied with other mtDNA sequence information. For trol should be employed with each sequencing run. example, since HVl and HV2 are genetically linked due to their mode of inheritance, it would not be appropriate to multiply the frequency of a mtDNA sequence from HVl Contamination with that of HV2. HVl and HV2 together constitute a single mtDNA type. Therefore, at this time the frequency of a The most critical potential source of error in mtDNA mtDNA type within the population is calculated by the sequencing is contamination. If more than one individual's counting method, or the number of times a particular se­ DNA is extracted and amplified, the sequencing results will quence has been observed in a population, divided by the reflect this mixture. In extreme cases, the contaminating number of sequences contained within the appropriate data­ DNA can greatly exceed the DNA from the donor and base. thereby yield a false positive result. This fact would not be In forensic casework, it may not always be possible to detected without the appropriate negative controls. Reagent amplify and/or sequence the entire regions defined as HVl blanks and negative controls should be employed with each and HV2. In some cases" the size of the amplified product extraction and amplification. Reagent blanks arise from an could be significantly smaller. In these cases, to obtain a extraction procedure with no added biological material. statistical estimate it is necessary to precisely define the • Sterile water or buffer is carried through the extraction starting and stopping points of the sequence to be queried protocol, and the resultant extract is amplified as normal. with respect to the database. For example, if data on only 100 This control tells the investigator if the extraction reagents base pairs within HVl from position 16100 to position 16200 are contaminated with human DNA. Negative controls ar.e are available, the database should be queried for a frequency amplification-specific and test whether amplification re­ only within these two points. The remaining information in agents are contaminated. Sterile water or buffer is added to the database should not be used for statistical purposes in a PCR reaction .in lieu of DNA and thermally cycled as the this particular case. amplification protocol dictates. As discussed previously, forensic sequence inform a tion The high sensitivity level of this method results in an might not be without ambiguity at certain positions. In such increased possibility of contamination, which necessitates cases, all four nucleotide frequencies in the reference popu­ additional controls. If possible, based on the amount of lation might be considered at each position of ambiguity. tissue available for extraction, separate portions of the tissue For example, consider the mtDNA sequence from positions should be separately extracted and amplified. This muIti­ 80 to 100 (HV2). The sequencp. from Anderson et al. (1981) pHcity gives the investigator an indication of the possibility follows: CGA TAG CAT TGC GAG ACG CTG. of contamination and serves the additional purpose of inde­ Suppose a forensic sample is amplified and sequenced pendently confirming the sequence information from an from positions 80 to 100. The forensic sample is sequenced independent extract of the same evidentiary material. In the in duplicate, but at position 86 the base cannot be deter­ case of a single evidentiary hair, however, dupHcate extrac­ mined. The forensic sample is also found to contain a tions might not be possible. Future research should be polymorphism with respect to the Anderson sequence at directed toward determining the minimum amount of tissue position number 98. Instead of a cytosine at this position, a necessary to obtain sufficient DNA for amplification. The thymidine is found. One way to search the mtDNA database sequencing results should be compared to the known mtDNA for such a sample might be to query for the frequency of the type of the donor (obtained from purified DNA from blood, following sequence beginning at position 80 and ending at for example) to show that the PCR-amplified amplicon from position 100: trace amounts of template exactly match that from more 80 100 • abundant sources of DNA template. CGA TAG NAT TGC GAG ACG TTG Volume 20 Number 4 October 1993 73 ~~ ~~ ~~~ ~~ .~~: a~~ ~ ~~! ~~I~ ~~~ ~~~~~; ~~ ~~~ n~ ~.. ~~! ~~ ~ ~~~ M§~ ~ M~Il;§ a~§ ~ ~~! ~~ ~I~ ~. ~T2- 'S, ;TITIG ITIT GIA IA AlA - [A'-I-'+"+"_'''_i!'-I-''-I-_++++-++'''+-:-.:"",f"·4 ~"+:'1---F~-+--t:--~-t:-fc'F+c--F+'+-+'4~-+=--~'-Ff~+'-R--FPlf--'Ft::-F+--t=-t;;" r.:-le_s'3;;T!J"~-----t·r+--r-t Ie ++-I---t--Fl-H-t-++-H--.-J-++·H-J-+-+-+++++--+·--t++--t+-t··HI---+---t IU~ H-i~H+i'i f§!Jf- Le-ST7 le-STB T ++++-t+iH-+++-++t--I-H- r.:-Iv=~~~_·~-++++--rt-t-r+~~~-t~-i·i+++-~t++~~+---t+·b-~+---t++-+-+-'H-f++-t-++t-.+-,~~+1~10 ~_ le-ST/1 If:ST/2 le-sTI' Ie-sTI , le-Si

Figure 3. Upon completion of sequence confirmation, multiple sequences can be aligned and presented together. This data is frol/l Stoneking et al. (1991). The standard sequence (Anderson) is shown on the first line. Deviations from tltis sequence are shown as tlte corresponding letter of the Ilucleotide 1uhich differs from the reference. Where the boxes are empty, tlte sequence of the sample matches that of the reference, Where a polymorphism with respect to the reference (Anderson) sequence exists, tlte differing nucleotide is placed

• 74 Crime Laboratory Digest ill the appropriate box. Trallsversiolls are prill ted in boldface. Horizontallines denote sequence illformation which was Ilot determined. Dl'/etiolls are represented by a dash. Notice tltat in some positions, such as nucleotide positions 263 alld 310.1 in HV2, the reference nucleotide is rare in tlte population, alld a better way to report tlte informatioll might be to establish a different reference sequence, olle wlliclt represents the most common base at each position .

•

Volume 20 Number 4 O('t~ber 1993 75 The N at position 86 tells the computer to allow any of DNA molecule or by additional sequencing of the same the four possibilities at this position. The T at position 98, in strand. addition to all the other positions, is fixed. Then, in effect the 4. A common nomencla ture for primer designa tion should computer sums the frequencies of the following sequences: be adopted. Presently, mtDNA primers are listed by the strand (H for heavy and L for light) followed by the • CGA TAG AAT TGC GAGACGITG numeric designation from Anderson et al. (1981) of the CGATAG ~AT TGC GAGACGITG 3' ending base of the primer. CGA TAG GAT TGC GAGACG TTG 5. Laboratories should share template for CGA TAG IAT TGC GAGACGITG interlaboratory comparison of sequence information. This will facilitate validation of numerous sequencing techniques, ensuring that quality control can be main­ Discussion tained regardless of a laboratory's methodology. Inde­ pendent, nonforensic laboratories should be included in Mitochondrial DNA sequencing is a viable technique this effort to implement consensus quality control mea­ for human identification testing. Its advantage over nuclear sures. DNA typing methods is the high copy number of mtDNA 6. A human mtDNA database from HV1 and HV2 of the over nuclear DNA and hence the added sensitivity in cases D-Ioop should be compiled from existing published where one is confronted with a limited amount of DNA or sequences and ongoing research. When feasible, DNA the DNA is significantly degraded. The maternal inherit­ samples from contributors of published mtDNA con­ ance of mtDNA offers the forensic scientist reference samples trol region sequences found in the literature should be from maternally related individuals which are, barring obtained and sequenced by forensic laboratories to mutation(s), identical. Nuclear markers from related indi­ ensure interlabora tory quality control and fidelity of the viduals share common alleles but are not genotypically forensic mtDNA database. identical. As with all forensic identification techniques, issues It should be stressed that mtDNA sequencing is an related to reliability and reproducibility must be addressed. analytical procedure which the forensic scientist must un­ Automated sequencers offer high throughput and ease of derstand in its proper context. In routine forensic cases da ta handling but also introduce issues related specifically to where nuclear DNA is extracted from a sample, polymor­ this technology. Replication of sequencing runs, which phic nuclear markers such as DQa., short tandem repeat loci, independently confirm nucleotide designations, is a good etc., can and should be utilized. These markers should take wny of overcoming sequence ambiguities. As many differ­ precedence over mtDNA analysis. Not only are such mark­ ent methods exist to sequence DNA, templates should be ers currently more statistically informative than mtDNA, shared among laboratories, and the results should be inde­ they also are not as susceptible to contamination as the pendently assessed for reliability. • analysis of mtDNA. However, mtDNA can and should be Validation studies must be conducted on any new the method routinely used with certain classes of biological forensic technique. As the chemistry of DNA is very well evidenc

Gyllensten, U. B. and Erlich, H. A. Generation of single­ Orrego, C. and King, M. C. Determination of familial stranded DNA by the polymerase chain reaction and its relationships. In: PCR Protocols: A Guide to Methods alld application to direct sequencing of the HLA-DQA locus, Applications, ed. M. A. Innis, D. H. Gelfand, J. J. Sninsky, Proceedings of the National Academy of Sciences of the United and T .J. White, 416-426. Academic Press, San Diego, CA, States of America (1988) 85:7652-7656. 1990.

Higuchi, R., von Beroldingen, C. H., Sensabaugh, G. F., and Sanger, F., Nicklen, S., and Coulson, A. R. DNA sequencing Erlich, H. A. DNA typing from single hairs, Nature with chain-terminating inhibitors, Proceedings of tlle (1988) 332:543-546. National Academy of Sciences of the United States ofAmerica (1977) 74:5463-5468. Holland, M. M., Fisher, D. 1., MitcheH, 1. G., Rodriguez, W. • C.,Canik,J.J., Merril, c.R., and Weedn, V. W. Mitochon­ Smith, 1. M., Sanders, J. Z., Kaiser, R. J., Highes, P., Dodd, drial DNA sequence analysis of human skeletal remains: c., Connell, C. R., Heiner, c., Kent, S. B. H., and Hood, Identification of remains from the Vietnam War, Journal L. E. Fluorescence detection in automated DNA se­ of Forensic Sciences (1993) 38:542-553. quence analysis, Nature (1986) 321:674-679.

Hopgood, R., Sullivan, K. M., and Gill, P. Strategies for Stoneking, M., Hedgecock, D., Higuchi, R. G., Vigilant, L., automated sequencing of human mitochondrial DNA and Erlich,H.A. Population variation of human mtDNA directly from PCR products, Biotec1miques (1992) 13:82- control region sequences detected by imzymatic amplifi­ 92. cation and sequence specific oligonucleotide probes, American Journal of Human Genetics (1991) 48:370-382. Horai, S., Kondo, R., Nakagawa-Hattori, Y., Hayashi, S., Sonoda, S., and Tajima, K. Peopling of the Americas, Sullivan, K. M., Hopgood, R., and Gill, P. Identification of founded by four major lineages of mitochondrial DNA, human remains by amplification and automated se­ Molecular Biology and Evolution (1993) 10:23-47. quencing of mitochondrial DNA, International Journal of Legal Medicine (1992) 105:83-86. Hutchinson, C. A., Newbold, J. E., Potter, S. S., and Edgell, M. H. Maternal inheritance of mammalian mitochon­ Vigilant, L. M., Stoneking, M., Harpending, H., Hawkes, drial DNA, Nature (1974) 251:536-538. K., and Wilson, A. C. African populations and the evolution of human mitochondrial DNA, Science (1991) 253:1503-1507. • Volume 20 Number 4 October 1993 77