Forensic DNA Typing of a Single Hair: Mitochondrial DNA Sequencing and Highly Discriminating STR Multiplexes Developed for Various Detection Platforms
Danuta Miscicka-Sliwka, Tomasz Grzybowski, Marcin Wozniak, and Jakub Czarny
Forensic Medicine Institute, The Ludwik Rydygier’s University School of Medical
Sciences, ul. M. Curie-Sklodowskiej 9, 85-094 Bydgoszcz, Poland
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INTRODUCTION
The application of DNA typing for identification of individuals provides objective evidence in the characterization of biological materials found at crime scenes worldwide. Throughout the past few years, the forensic identity testing has been considerably transformed by the development of multiplex PCR systems to investigate highly polymorphic short tandem repeat (STR) loci (1-3), offering both higher throughput and greatly increased sensitivity over conventional DNA single- and multilocus probe techniques. Due to their advantages of definitive allelic assignment, amenability to automation on DNA sequencers and compatibility with multi-color detection platforms, highly discriminating multiplex STR systems are ideally suited for both high throughput forensic identification and the formation of large national DNA databases for criminal intelligence purposes (4-8).
Although nuclear markers are very accurate for determining human identity, there are certain cases for which they can not be used and mitochondrial DNA (mtDNA) testing can serve as an alternative. Because mtDNA is circular, it is less susceptible to exonuclease degradation. Thus, mtDNA testing provides an efficient tool for the analysis of severely degraded human remains (9,10). Moreover, mtDNA is present in 1,000 to 10,000 copies per mammalian cell, being extremely useful genetic marker when only a minute amount of DNA is available. In addition to its high copy number, the other characteristics that makes mtDNA useful for human identification are its maternal mode of inheritance and high level of polymorphism of non-coding D-loop region. Consequently, DNA amplification and automated sequencing of the mt D-loop region complements the STR approach in a large number of DNA typing laboratories (including our Institute).
One of the important applications of both fluorescent STR multiplexes and mtDNA sequencing in forensic casework is the analysis of hair. It is noteworthy that hair samples found at a crime scene may represent the only available biological evidence. However, DNA typing from single hair is often problematic in forensics since (i) individual hairs contain very small quantities of DNA and (ii) the evidentiary hair samples often have roots of bad quality or even no roots at all.
The aim of this experimental study is to optimize PCR-based DNA typing of single hairs by means of DNA extraction, quantitation and amplification efficiency obtained using highly discriminating multiplex STR systems combined with two different detection platforms: a hexaplex PCR developed and validated in our laboratory (3,7) (Amelogenin, D1S103, TH01, D21S11, D18S51, FGA; labeled with fluorescein, visualized using a single-wavelength A.L.F. DNA Sequencer) and the GenePrint
Ô PowerPlexÔ from Promega Corporation (11) recently introduced to our laboratory (labeled with fluorescein and carboxy-tetramethylrhodamine, visualized using the ABI PRISMÔ 377 DNA Sequencer). Additionally, DNA sequencing of both HV1 and HV2 segments of the mitochondrial non-coding region was performed as an important complement to STR-based DNA typing of individual hairs in forensics.MATERIALS AND METHODS
DNA Extraction and Quantitation
DNA from 20 single hair roots and 10 hair shafts obtained from freshly-plucked hairs of unrelated individuals was isolated by digestion with proteinase K at 56
°C overnight (extraction buffer contained 10 mM Tris, 100 mM NaCl, 39 mM DTT, 10 mM EDTA and 2% SDS) followed by a three-step organic extraction procedure involving phenol: chloroform: isoamyl alcohol and chloroform, with an additional chloroform extraction. DNA samples were then subjected to ultrafiltration with Microcon-100 microconcentrators (Amicon) and quantified by dot blot hybridization to a probe for a specific human alpha satellite sequence (D17Z1) using the QuantiBlot kit (Perkin Elmer). Simultaneously, quantitation of samples was being performed spectrophotometrically using a GeneQuant DNA Calculator (Pharmacia Biotech).Multiplex Amplification Conditions
A hexaplex PCR amplification was performed using 0.5-10 ng of genomic DNA in a 25
ml reaction volume comprising 1X Promega buffer; 1.5 mM MgCl2; 200 mM of each of the dNTPs (Promega); 0.625 U of Taq polymerase (Promega); 0.4 mM of each HUMD1S103 primer; 0.14 mM of each Amelogenin primer; 0.2 mM of each HUMTH01 primer; 0.25 mM of each HUMD21S11 primer; 0.14 mM of each HUMD18S51 primer; 0.2 mM of each HUMFIBRA primer. One primer per pair was 5’-fluorescein labeled. Amelogenin, HUMTH01, HUMD21S11 and D18S51 primer sequences were used according to Oldroyd et al. (12). HUMD1S103 and HUMFIBRA primer sequences were designed with OLIGO v.5.0 software and published previously (7).After a hot start at 94
°C for 2 minutes, samples were amplified for 30 cycles of 30 seconds at 93°C, 75 seconds at 58°C and 15 seconds at 72°C followed by a final 15 seconds extension at 72°C (Perkin-Elmer Thermal Cycler 9600).The GenePrint
Ô PowerPlexÔ and Amelogenin amplification was performed using 0.5-5 ng of genomic DNA according to the manufacturer’s protocol.Detection Platforms
PCR products of the hexaplex were separated in a 5.7% denaturing polyacrylamide (8M urea) gel and sized automatically on an A.L.F. DNA Sequencer (Pharmacia Biotech). The external size standard used was Sizer 50-500 bp (Pharmacia Biotech) loaded into every fifth gel lane. The internal size standard was the shorter product of the Amelogenin locus (106 bp). Data were collected using A.L.F. Manager software (Pharmacia Biotech) running on an OS2 v. 2.1 platform. Fragment lengths were analyzed using Fragment Manager software (Pharmacia Biotech).
The PowerPlex
Ô PCR products were separated in a 5% Long Ranger acrylamide gel (7M urea) on the ABI PRISMÔ 377 DNA Sequencer. The internal standard used was the Fluorescent Ladder, 60-400 Bases, labeled with carboxy-X-rhodamine (Promega). Fragment sizes were determined automatically using Genescan v. 2.1 software (Perkin-Elmer).Mitochondrial DNA Amplification and Sequencing
Mitochondrial PCR amplification of the entire non-coding region (30 cycles) was performed using 20-80 ng (hair roots) or 0.5-1 ng of total genomic DNA (hair shafts) in a 25
ml reaction volume comprising 1X Promega buffer; 1.5 mM MgCl2; 200 mM of each of the dNTPs (Promega); 1.5 U of Taq polymerase (Promega) and 1 mM. primers L15926 and H00580. Each cycle comprised 20 seconds at 94°C, 30 seconds at 50°C and 2.5 minutes at 72°C (Perkin-Elmer Thermal Cycler 9600). The resultant D-loop amplification product from hair roots was diluted 1000-fold and 4 ml aliquots were added to an array of second round, nested PCR reactions (32 cycles) to generate sufficient DNA templates for sequencing, using the conditions given above, except for the reaction volume was 50 ml, extension time was 1.5 minutes, enzyme concentration was 1 U and the primer concentrations were 0.1mM. For hair shafts, nested PCR amplifications were performed using aliquots of first PCR mixture 0.5 ml (not diluted). To generate templates for sequencing both strands of the hypervariable segment HV1, the primer sets L15997/M13(-21)H16401 and M13(-21)L15997/H16401 were used in separate amplification reactions. Similarly, sequencing templates of the segment HV2 were generated in two PCR reactions using primer sets L00029/M13(-21)H00408 and H00408/M13(-21)L00029, respectively. Both primer sequences and nomenclature were used according to Sullivan et al. (13).PCR products were purified by ultrafiltration (Microcon 100, Amicon) and sequenced directly with (-21)M13 primer using Dye Primer Cycle Sequencing Kit (Perkin Elmer) according to the manufacturer’s protocol. Sequencing products were separated in a 4% denaturing polyacrylamide gel on the ABI PRISMÔ 377 DNA Sequencer. Data was analyzed automatically using DNA Sequencing Analysis software (Perkin-Elmer).
RESULTS AND DISCUSSION
DNA Extraction: Single-step Vs. Three-step Protocol
The standard organic extraction method used in this study removes proteins and other cellular components from nucleic acids, providing relatively purified DNA preparations. Moreover, the ultrafiltration step enables both concentrating the samples with small amounts of DNA and removing low molecular weight contaminants that would affect the ability to amplify target DNA or result in an unacceptable level of artifactual peaks. Interestingly, the DNA extraction procedure appeared to be one of the most critical parameters for both hexaplex and Powerplex amplifications. Initially, a single-step organic extraction procedure involving phenol: chloroform: isoamyl alcohol had been used. This resulted in an increased non-specific background amplification in the hexaplex reactions, with the enormous TH01 product yields. Similarly, a large number of "stutter" peaks was observed at those Powerplex loci which were labeled with TMR (CSF1PO, TPOX, TH01, vWA). These undesirable effects were avoided (Fig.1) by the use of a three-step procedure, with an additional double chloroform extraction resulting in removal of any lingering traces of phenol from the nucleic acid preparation. Furthermore, it is possible that subsequent extraction with chloroform removed other contaminants responsible for the occurrence of amplification artifacts. It is noteworthy that it is unnecessary to perform a three-step DNA extraction for the mtDNA sequencing purposes, since mtDNA amplification was not influenced by the use of a single-step protocol.
DNA Quantitation
Accurate quantitation of human DNA is particularly important for multiplex STR amplifications where optimal results are obtained using a relatively narrow range of template DNA concentration. In this study, DNA samples extracted from single hairs were quantitated using both slot blot hybridization and spectrophotometric approaches. A wide variation of the DNA amount in hair roots from different individuals was observed. As measured by slot blot quantitation, the root end of a single hair contained between 20 and 300 ng DNA, with 150-200 ng being the average, whereas hair shafts contained as little as 1-1.5 ng. It clearly corresponds to the early report of Higuchi et al. (14). Surprisingly, according to the spectrophotometric determination of the amount of DNA, the roots of single hairs contained as much as 10 mg DNA! The only possible explanation of this phenomenon is that the samples contained significant quantities of impurities (i.e. melanin) that interfered in the spectrophotometric measurement of the amount of ultraviolet irradiation absorbed by the bases. Thus, the spectrophotometric approach appeared to be completely useless in determination of the amount of DNA extracted from hair.
Multiplex Amplification of the DNA Obtained from Single Hairs
Since the DNA content of evidentiary hairs is usually limited and/or degraded, it is particularly desirable to carefully evaluate any multiplex STR system in terms of amplification sensitivity obtained from both single hair roots and hair shafts. Also, the effect of potential inhibitors to the reaction should be considered. For single hair roots, template DNA concentration was tested over a range between 0.5 and 10 ng for hexaplex reactions performed in a 25
ml volume. The optimal amount of nuclear DNA required for hexaplex amplification was from 2 to 5 ng. The reduction of template DNA concentration to 0.5-1 ng resulted in reduced product yields for all 6 loci within the hexaplex. Locus drop-out was particularly pronounced for TH01. At 10 ng and higher DNA concentrations, D21S11 and D18S51 product yields continued to increase, although overamplifications did not display any nonspecific bands. The above results are summarized in Fig.2.The GenePrint
Ô PowerPlexÔ system turn out to be more sensitive than the hexaplex. As little as 500 pg of template DNA obtained from single hair root was successfully amplified and the correct allelic designation obtained. Concentration of 1 ng appeared to be optimal (Fig.3), whereas amplification of greater than 1.5 ng of template DNA resulted in an occurrence of additional peaks in the Fluorescent Ladder (Fig.4). Those peaks were due to the overamplification of the Powerplex loci. Thus, the Powerplex was not only more sensitive than the hexaplex developed for a single-wavelength DNA sequencer, but also less tolerant to changes in template DNA concentrations.A number of publications report that highly discriminating multiplex systems are generally less sensitive than single locus reactions since the conditions used are a compromise (1,6,7,15). However, for both multiplex systems, the level of sensitivity was sufficient enough to obtain full profile amplifications from the DNA obtained from single hair shafts (1-1.5 ng of the DNA recovered). Hexaplex reactions performed with 0.5-1 ng of DNA template gave full profiles, although the product yields were significantly reduced. Nevertheless, sensitivity was further increased by increasing the individual primer concentrations (up to 20%). No allelic drop-out was observed at low template levels. The PowerPlex™ amplifications gave satisfactory signal even from 500 pg of DNA, so DNA typing from single hair shafts was straightforward using this system.
As a result of Microcon-100 ultrafiltration, any inhibition effect was observed at higher DNA concentrations using both multiplexes.
Mitochondrial DNA Sequencing
As mentioned earlier, mitochondrial sequences are present in multiple copies per cell and can consequently be amplified in critical situations, where nuclear markers often fail to work. Moreover, polymorphic information is limited to two small regions, which can be automatically sequenced in a single electrophoresis lane using a fluorescently labeled universal sequencing primer. In our study, amplification products of the entire D-loop region (1333 bp) were obtained from both the root and shaft portions of the hairs. Routinely, if there was a sufficient amount of DNA available (hair roots), mitochondrial PCR amplification of the entire non-coding region was performed using 20-80 ng of total genomic DNA and 4
ml aliquots of the resultant, 1000-fold diluted D-loop product were added to an array of second round, nested PCR reactions to generate DNA templates for sequencing. However, when the amount of sample DNA was very limited (hair shafts), the initial D-loop region amplification was carried out with 0.5 ng of genomic DNA and large excess of specific primers. Next a second nested reaction was performed using aliquots of PCR mixture 0.5 ml containing the resultant 1333 bp amplification product (not diluted).This approach took several advantages of the nested PCR amplification. Since (i) nested PCR is extremely sensitive, the initial amplification provides enough material for multiple second amplifications, making this a safe protocol to use when the DNA amount recovered from single hair shafts is very limited. Moreover, (ii) due to the additional selectivity of the second round of amplification, nested PCR usually generates a cleaner PCR product than direct PCR leading to cleaner sequencing results. In the case of some particularly difficult hair shafts, slight modifications of the protocol described were necessary to introduce in order to increase the product yields. First, primer concentrations in the second round of amplification were doubled. Since the excess of PCR primers was removed by subsequent Microcon 100 ultrafiltration, this modification did not affect sequencing reactions. Second, the cycle number of nested PCRs was increased to 35. Similarly, it did not seem to influence sequencing reactions as long as non-specific products appeared.
It is noteworthy that the dye primer cycle sequencing approach we routinely use for mtDNA sequencing is robust and relatively tolerant of variations in the amount of template DNA added to the reaction (it typically consumes as little as 100 ng of sequencing template), being extremely advantageous when the nested amplification efficiency is slightly reduced. Figures 5 and 6 show typical sequencing data obtained from both single hair root and hair shaft. Even though the use of Sequenase typically produces more even peak intensities (16,17), the dye primer cycle sequencing chemistry yields data with relatively low background noise enabling reliable detection of subtle sequence features such as heteroplasmy.
CONCLUDING REMARKS
It is obvious that PCR-based typing techniques offer several advantages compared with RFLP technology, such as increased robustness and sensitivity. The latter feature is critical in analyzing minimal amounts of DNA found in single hairs. According to our results, even the shaft portions of the hairs contain enough copies of both nuclear and mitochondrial DNA to be detected using PCR. For all hair shafts investigated, the amount of DNA extracted was sufficient enough not only to generate clean sequence data from both mtDNA hypervariable segments, but also to obtain full profiles using both multiplex STR systems employed. As expected, the most critical parameter in successful genetic analysis of a single hair was accurate quantitation of human DNA. Since multiplex STR systems appear to be extremely sensitive, the number of different genetic marker tests can be maximized using the smallest volume of DNA extract necessary for successful amplification.
Employing highly polymorphic multiplexes allows not only increase in sensitivity but also increase in the power of discrimination of the analysis. Consequently, the multiplex STR approach may be the last word in forensic DNA typing of single hair. It is noteworthy, however that the aim of this study was to provide some standard values in extraction, quantitation and amplification of genetic material derived from individual, freshly-plucked hairs. Since the hairs found at the crime scene are usually shed hairs, the success rate for actual casework can be substantially lower. Thus, depending on the quantity of recoverable DNA from particular hair sample, a decision should be made to type the sample by the more informative, but currently less sensitive STR approach or by the less discriminating but more sensitive mtDNA sequencing.
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Figure 1. An electropherogram of hexaplex STR profiles obtained from a single hair root using 5ng of total genomic DNA. The top panel shows PCR amplification affected by the use of a single-step DNA extraction procedure involving phenol: chloroform: isoamyl alcohol. Both non-specific background amplification and the enormous TH01 product yields can be observed. After the use of a three-step extraction procedure (with an additional chloroform extraction), clean STR profile was obtained (the bottom panel).
Figure 2. An electropherogram of a hexaplex PCR amplification performed with varying template concentrations. The optimal amount of nuclear DNA required for hexaplex amplification from a single hair root was between 2 and 5 ng (lanes 11 and 19). The reduction of template DNA concentration to 0.5-1 ng resulted in reduced product yields for all 6 loci within the hexaplex (lanes 1 and 2). At 10 ng DNA concentration, D21S11 and D18S51 product yields continued to increase, although overamplifications did not display any nonspecific bands (lane 28).
Figure 3. The GenePrint™ PowerPlex™ STR System and Amelogenin (Promega) applied to the genetic analysis of a single hair. As little as 500 pg of template DNA obtained from a single hair root was successfully amplified at nine loci (the top panel). The bottom panel shows an optimal result achieved using a 1 ng genomic DNA sample recovered from single hair roots.
Figure 4. The GenePrint™ PowerPlex™ profile obtained from a single hair root using 2 ng of total genomic DNA. Additional peaks in the Flourescent Ladder (marked red) appeared due to the slight overamplification of the PowerPlex™ loci.
Figure 5. Typical mitochondrial DNA sequencing data obtained from a single hair root. The PCR fragment is from HV1 and was sequenced using dye primer cycle sequencing chemistry.
Figure 6. Sequencing data of the mtDNA HV2 region obtained from a single hair root.
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