J Forensic Sci, March 2015, Vol. 60, No. 2 doi: 10.1111/1556-4029.12682 Available online at: onlinelibrary.wiley.com

TECHNICAL NOTE CRIMINALISICS

Byron C. Smith,1 M.S.F.S.; Emily Vandegrift,2 M.S.F.S.; Valerie Mattimore Fuller,3 Ph.D.; Robert W. Allen,4 Ph.D.; and School of Forensic Sciences

Evaluation of Degradation in DNA from Males with a Quantitative Gender Typing, Endpoint PCR Multiplex*

ABSTRACT: Evidentiary samples submitted to a forensic DNA laboratory occasionally yield DNA that is degraded. Samples of intact chro-

mosomal DNA (both nuclear and mitochondrial) were subjected to a heating protocol to induce DNA degradation. The DNAs were then analyzed using a multiplex PCR assay that amplifies targets of low and high molecular weight on the X/Y and mitochondrial chromosomes. If degradation is random, the amplification of larger DNA targets should be more adversely affected by degradation than smaller targets. In nuclear and mitochondrial DNA from a male donor, exhibiting degradation, DNA quantity estimates based upon higher molecular weight amplicons (HMW) are significantly lower than estimates made using low molecular weight (LMW) Q-TAT amplicons. DNA degradation estimated using this approach correlated well with actual fluorescence associated with HMW and LMW STR alleles amplified from the same genomic DNA templates. Q-TAT is thus useful not only as a quantitation tool, but also as an indicator of template degradation.

KEYWORDS: forensic science, DNA degradation, endpoint PCR, amelogenin, SRY, quantitative fluorescence, Q-TAT assay Situations in the forensic DNA laboratory that can compromise the quality of STR profiles produced from evidentiary samples include an insufficient quantity of template recovered for PCR, the presence of PCR inhibitors co-extracted with the DNA, and DNA template that is partially or wholly degraded due to the type of evidentiary sample submitted. Degradation, in particular, is a common problem encountered by laboratories that process human remains for identification purposes. Since the beginning of the last decade, and brought into sharp focus by the 2001, World Trade Center attack, there has been an emphasis around the world to effectively identify human remains through DNA testing. Political and/or social events in some countries have created large numbers of unidentified individuals, and in many cases, these remains have existed in clandestine graves for many years. Through testing DNA recovered from these remains and comparing results with DNA profiles from surviving family members, there is a reasonable chance of identifying the victim and reuniting his/her remains with family.

1 Forensic Laboratory, Tulsa Police Department, 1111 W. 17th Street, Building E, 2nd Floor, Tulsa, OK. 2 Tennessee Department of Public Safety, Nashville, TN. 3 National Forensic Science Laboratory, Ministry of Legal Affairs, Government of Saint Lucia, Tapion Castries, West Indies. 4 School of Forensic Sciences, Oklahoma State University, 1111 West 17th Street, Tulsa, OK. *Supported, in part, by the H.A. and Mary K. Chapman Charitable Trust and a grant from the Midwest Forensic Resource Center (MFRC), Ames, Iowa. Disclaimer: Robert W. Allen, PhD, and Valerie Mattimore Fuller (25), PhD, are the holders of a patent describing the Q-TAT assay utilized in this study. Received 24 May 2013; and in revised form 24 Jan. 2014; accepted 18 Mar. 2014.

© 2014 American Academy of Forensic Sciences

Molecular methods to assess the integrity of DNA recovered from a sample after extraction, but before STR amplification and analysis have been described, and involve both real-time PCR and endpoint PCR analysis strategies (1–5). In most cases, including capillary electrophoresis-based methods, the fluorescence from PCR amplicons of differing size can be compared. For the purposes of DNA profiling, the integrity of DNA template must be sufficient to successfully amplify STR alleles ranging in size from 100 to ~350 base pairs. If the fluorescence from an amplicon of relative large size (~200–300 base pairs) is compared with that in an amplicon of small size (~100 base pairs), the ratio of fluorescence between the two products amplified from a DNA template that does not exhibit extensive degradation can be determined and should be around 1.0. In a degraded sample, however, the ratio of fluorescence in the amplicons of different size may deviate from a ratio of 1.0. The nature and degree of the ratio deviation might be informative as to just how degraded the DNA template could be (1–5). A postamplification, DNA quantitation assay (Q-TAT) was described in several publications (6–10). The Q-TAT assay incorporates a multiplex PCR cocktail that includes primers directed against the amelogenin locus (Amel-X/Y; 210 and 216 base pairs, respectively), primers directed against the SRY gene (110 base pair amplicon) (11,12), and primers directed to a portion of the hypoxanthine phosphoribosyl transferase gene (HPX; 99 base pairs); all targets reside in nuclear DNA (nDNA). The Q-TAT assay can also incorporate additional sets of primers directed against two DNA targets in mitochondrial DNA (mtDNA), located in the region of the origin of replication (mt97 and mt287) (13). The Q-TAT assay has been validated in several practicing forensic laboratories and has proven reliable as a quantitation method for total human and male nuclear DNA 399

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(7–9). The assay also includes primers and a synthetic DNA template that serves to detect inhibitors that may exist in a DNA extract (7). Given the differences in size of the Q-TAT amplicons, we investigated the potential of the assay to also assess the integrity of a nuclear (nDNA) and mitochondrial DNA (mtDNA). Intact chromosomal DNA isolated from a male (i.e., DNA template capable of directing the amplification of a complete STR profile with multiplex STR typing kits that exhibits balanced peak heights within and between STR loci of all sizes) was extracted from freshly procured blood and was subjected to controlled degradation that was confirmed by agarose gel electrophoresis. In the Q-TAT assay, the ratio of the DNA quantity estimate based upon fluorescence associated with the SRY amplicon versus the DNA quantity estimate based upon fluorescence associated with the Amel-X+Y amplicons was evaluated in each sample as a potential reflection of the extent of DNA template degradation. In other experiments, Q-TAT amplicon ratios were correlated with STR typing results for commonly used STR kits. Our results support the utility of the Q-TAT assay to not only quantify DNA recovered from a male sample, but to also predict the state of integrity of DNA templates extracted from forensic samples in a way that is applicable for deciding how best to proceed with DNA testing. The assay is compatible with standard instrumentation typical of a forensic DNA laboratory (i.e., a GeneAmp PCR System 9700 thermal cycler and either a 310, 3130, or 3130xl Genetic Analyzer) (Life Technologies Inc., Foster City, CA), or even with polyacrylamide gel platforms such as the FMBIO (Paternity Testing Corporation, Columbia, MO, personal communication). The Q-TAT assay may be especially well suited for small laboratories that face working with challenging samples (8,10).

Materials and Methods Q-TAT Assay The basic elements of the Q-TAT assay have been described in earlier publications (6,7). Enhancements to the assay, since 2010, include an additional primer set directed against a small portion of the hypoxanthine phosphoribosyl transferase gene located on the X chromosome (99 base pair product, HPX). The HPX marker represents a second PCR product produced from the X chromosome that is small in size much like the SRY amplicon. Another enhancement to the assay is the optional inclusion of primers targeting two regions of the D loop in mtDNA that are in close proximity to the origin of replication (13). The 97 bp product (mt97) encompasses nucleotides 37 through 133 in the mitochondrial genome, and the mt287 product (mt287) encompasses nucleotides 16113 through 16382 (13). The mt97 and mt287 products are labeled with the NED fluorescent dye to distinguish them from the products produced from nDNA which are labeled with 6-FAM fluorescent dye (7). The Q-TAT multiplex also incorporates an added recombinant plasmid (pRL) harboring the luciferase gene from a marine coelenterate known as the sea pansy, which serves as an indicator of PCR inhibition (as it is included in the master mix added to DNA samples being quantified) (7). Template DNA quantity is estimated in the Q-TAT assay using a standard curve that contains a dilution series from a male standard genomic sample of known quantity (6–9). Initially, twofold serial dilutions of standard DNA (either prepared in-house from fresh blood, or purchased as a NIST quantification

standard) were produced from 1000 pg down to 32.125 pg, and each sample was amplified using Q-TAT. It should be noted that standard curves can also be effectively produced using threefold serial dilutions of the 1000 pg/lL sample, much like the recommended process used with Quantifiler qPCR kits (Life Technologies Inc.). Fluorescence, expressed as either peak height or area, was captured using GeneMapper ID software (ver. 3.2, Life Technologies Inc.) and plotted to produce the curve. Excel was used to create macros to automate the entire process for estimating the DNA concentration in any sample, and the entire Q-TAT process can be completed in the same basic time frame as qPCR (~2 h)(unpublished observations). Extraction of Genomic DNA from Blood Freshly procured blood was obtained from a male donor and was mixed with a five volume excess of TE4 to promote hemolysis. Following centrifugation at 500 9 g for 5 min at room temperature, the white cell pellet was resuspended with 0.5% SDS and 400 lg/mL proteinase K in 10 mM TRIS-Cl (pH 8.0) containing 0.2 M NaCl. DNA was extracted during incubation at 65°C for 1.5 h. After digestion, the sample was purified with phenol:CHCl3:isoamyl alcohol (9:0.96:0.04) extraction. DNA was precipitated with two volumes of 95% ethanol and captured using a disposable inoculating loop before resuspending in TE4. The DNA was quantified spectrophotometrically and also by yield gel to confirm the concentration estimate. This DNA migrated as a single band comparable to intact lambda bacteriophage DNA used as a quantitation standard. Degradation of DNA To degrade genomic DNA, the DNA isolated from a male donor’s liquid blood was dispensed into multiple 200-lL sample tubes and heated at 95°C in ultrapure water at 40–52.4 ng/lL in a GeneAmp PCR System 9700 thermal cycler (Life Technologies Inc.) (procedure suggested by Bruce McCord, Florida International University, personal communication). Before heating was initiated, and at varying intervals after reaching 95°C, samples were removed from the heating block and quenched either by placing the heated sample tube in a chilled 96-well metal block, or by adding 175 lL of ice cold TE-4 to the sample, which was then kept cold and amplified using Q-TAT, the Profiler Plus or Identifiler STR multiplex (Life Technologies Inc.) or the Powerplex 16 Hot Start multiplex (Promega Corp., Madison, WI). Amplifications using STR kits contained 500– 1000 pg/lL of unheated or heated DNA according to the volume and cycling conditions recommended by the STR kit manufacturer (Life Technologies Inc. and Promega Corp.). Data Analysis The analysis of the state of degradation of a DNA template is based upon the fluorescence contained within the high and low molecular weight amplicons produced with the Q-TAT multiplex. Total DNA quantity in a sample of unknown concentration was estimated from the fluorescence associated with the Amel-X+Y amplicons, applied to the standard curve prepared using a standard genomic DNA sample isolated from a male donor. As all samples included in this study were of male origin, the total male-only DNA quantity could also be estimated from fluorescence associated with the SRY amplicon in a sample of unknown concentration using the same approach

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and with the use of the SRY standard curve (7,9). Thus, DNA quantity estimates in a sample could be made using a high molecular weight amplicons (HMW; Amel-X+Y) and also using a low molecular weight amplicon (LMW; SRY), and a ratio of the quantity estimates could be investigated as an indicator of degradation. This same approach was applied to the analysis of mtDNA co-extracted with the nDNA from the standardized male samples. Mt97 and mt287 represented the LMW and HMW amplicons, respectively, and the mt287 amplicon amplified from standardized DNA produced acceptable standard curves for quantifying mtDNA in extracts (13). Standard curves were linear up to 250 pg of input nDNA and were near linear up to 1000 pg of input nDNA. Worth mentioning is the fact that when working with mtDNA, one cannot express quantities of mtDNA in picograms as the standard curve for mtDNA was created using mtDNA co-extracted with nDNA in the DNA standard obtained from male blood. Therefore, a constant was incorporated into the mtDNA estimation procedure that assumes the typical blood cell contains about 500 mitochondria. It can be calculated that >99.99% of genomic DNA extracted from blood is nDNA (based upon the relative size of mtDNA versus nDNA and assuming there are 500 copies of mtDNA per white cell). Fluorescence in the mt287 product in an unknown could thus be plotted on the standard curve generated from fluorescence in the mt287 product amplified from the standard DNA. Where the fluorescence from the unknown fell on the curve, the quantity of predominant nDNA in that sample could be determined. That amount of nDNA divided by six (assuming 6 pg of genomic DNA/human cell) and then multiplied by 500, results in an estimate of the number of cell equivalents of mtDNA present in the sample. To effectively use the Q-TAT assay to quantify mtDNA, one need only know how many cell equivalents of mtDNA are required to produce a usable nucleotide sequence for mtDNA typing.

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Statistical Analyses Repeated Q-TAT analyses of DNA subjected to the degradation regimen were analyzed using one-way ANOVA with Tukey’s multiple comparisons test (eleven replicates). Tests of significance were performed for ratios of the DNA quantity estimates predicted using the SRY standard curve (LMW amplicon) versus the DNA quantity estimates predicted using the Amel-X/Y standard curve (HMW amplicons) as a measure of template degradation. In addition, one-way ANOVA with Tukey’s multiple comparison test was used to investigate the apparent correlation between the quantitative ratio from LMW/HMW amplicons in the Q-TAT assay and the ratio of fluorescence associated with STR alleles amplified from the D3S1358 locus (LMW products) versus those STR alleles amplified from the D16S539 locus (HMW products); both loci were amplified using the Identifiler multiplex kit (Life Technologies Inc.). Results The basic electropherogram produced with the Q-TAT multiplex is shown in Fig. 1. Amplicon products labeled with the 6-FAM dye are all produced from nuclear DNA (nDNA) templates with the exception of the pRL product at 200 bp, which is amplified from a recombinant plasmid harboring the luciferase gene from Renilla rentiformis (also known as the sea pansy). Reduced fluorescence associated with the pRL amplicon serves to detect PCR inhibitors (7). Fluorescence associated with the Amel-X+Y amplicons reflects total human DNA present and fluorescence associated with the SRY amplicon can also be used to estimate male-only DNA in a sample (7). In an intact DNA sample from a male, the picogram quantity estimates produced using SRY or Amel-X+Y should be close to one another. Indeed, in the report by Wilson et al. (7), this was shown to be the case. It should be noted that we have also demonstrated that

Fig. 1––Typical electropherogram produced from the Q-TAT assay. 500 picograms of “intact” DNA from a male reference standard was amplified in the QTAT assay and electrophoresed by capillary electrophoresis. The amplicons are labeled with both peak height and area in either the blue (6-FAM) or black (NED) dye channels. Amplicon size is shown on the top axis of the electropherogram.

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the HPX product (amplified from the X chromosome) can also be used to quantify the amount of DNA in a sample (not shown). The PCR products mt97 and mt287 are labeled with the NED dye and are amplified from the mitochondrial genome in the region of the origin of replication between HVI and extending slightly into HVII for mt97 and mt287, respectively. Standard curves for DNA quantitation were produced using a standardized, well-characterized DNA from a male. Curves for total and male DNA content were prepared using the Amel-X+Y or SRY amplicons, respectively (Fig. 2). The dynamic range for the assay spans 37–1000 pg, a range within which the standard curve fits a linear model well. In addition, R2 values above 0.99 are routinely possible, and routine use of an “average standard curve” produced from replicate analyses can be used repeatedly for different quantitation runs as long as internal standards of known concentration are included with each run and quantify as expected (Tulsa Police Laboratory, personal communication). Also shown in Fig. 2 is the linear portion of the standard curve produced from the mt287 product. The mt287 curve begins to deviate from linearity above 250 pg of input nDNA, a not surprising result given the molar excess of mtDNA template versus nDNA template extracted from a cell. The standard curve for the mt97 product deviates even more from linearity and at a lower input level of nDNA (not shown). Therefore, mtDNA quantitations were performed using mt287. It should be noted that R2 values in excess of 0.95 were routinely achievable for standard curves incorporating RFU for mt287 product with inclusion of the 500 and 1000 pg data points in the curve. As was discussed previously, the mtDNA estimates must be made in cell equivalents of mitochondrial DNA as a purified mtDNA sample is unavailable to serve as a standard.

Assessment of DNA Degradation Using Q-TAT Early attempts to degrade human DNA in a controlled fashion through the use of DNase 1 proved unsatisfactory for our purposes, primarily because DNase treatment consistently failed to eliminate all amplifiable genomic DNA template, even with extended incubations (unpublished observations). However, heating low concentrations of DNA in water at 95°C in a thermal cycler proved effective and controllable for degrading DNA in a way that produced the expected “smearing” of ethidium bromide staining in samples separated by size using agarose gel electrophoresis. Longer exposure to high temperature further reduced the size of the largest DNA fragments visible in a gel track and eventually degraded DNA to the point where neither Q-TAT amplicons nor STR amplicons could be produced using PCR (not shown). Confirmation that heating DNA at 95°C for varying lengths of time initiated degradation was obtained by electrophoresing aliquots of male genomic DNA using a 0.8–1.0% agarose gel subsequently stained with ethidium bromide (Fig. 3). Nonheated genomic DNA migrates as a single, high molecular weight entity similar to the migration of linear lambda DNA (~43 kb in size). This pattern is consistent with a high molecular weight sample that is large enough to produce a complete and balanced STR profile (not shown). Within the first 10 min of heating at 95°C, however, the single entity changes to a smearing of smaller DNA fragments, which progresses over time until a collection of fragments in the 100–400 bp range is all that remains after 90 min of heating (Fig. 3). The integrity of standard genomic DNA from a male was examined with the Q-TAT assay at selected time points during the 90-min duration of heating at 95°C (Table 1). Fluorescence associated with the Amel-X/Y amplicons begins disappearing

Fig. 2––Standard curves produced using the Amel-X/Y, SRY, or mt287 amplicons. Differing amounts of male reference DNA (abscissa) were amplified with Q-TAT. Fluorescence (area) under the peaks was plotted versus DNA amount. The mt287 amplicon was produced from the indicated amount of total DNA from a male that was initially extracted from a fresh blood sample.

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TABLE 1––RFU associated with Q-TAT amplicons produced from heated reference DNA samples. Nuclear DNA (Peak Height; RFU) Heat (min) 0 60 90

Amel-X/Y

% of Time 0

SRY

% of Time 0

5156 2368 862

100 46 17

2367 1534 809

100 65 34

Mitochondrial DNA (Peak Height; RFU)

Fig. 3––Agarose electrophoresis of reference DNA extracted from a male and subjected to 95°C heating for increasing 10-min intervals. Intact male reference DNA was dispensed into a series of 200-lL-thin-walled PCR tubes and heated to 95°C in a thermal cycler as described in Materials and Methods. Tubes were removed from the heat at increasing 10-min intervals (shown as numbers along the top axis of the electropherogram) beginning with a time zero sample (Lane 2). Maximum heating was for 90 min (Lane 11). Size standards are shown in lanes 1 and 12.

within 20–30 min of heating and decreases to 20–30% of the starting level after 90 min (Fig. 4a,b, Table 1). After 60–90 min of heating at 95°C, STR profiles produced with the Profiler Plus or PowerPlex 16 Hot Start (PP16HS) multiplexes exhibit a profile with fluorescence in high molecular weight alleles (e.g., D18S51 in the Profiler Plus kit, or D16S539 in the PowerPlex 16 Hot Start kit) being greatly diminished compared with fluorescence in low molecular weight alleles (e.g., D8S1179 in the Profiler Plus kit, or D5S818 in PowerPlex 16 Hot Start) (Fig. 4b). In fact, alleles for the CSF1P0 and Penta D loci (higher in molecular weight than D16S539) drop out of the PP16HS electropherogram altogether in products amplified from a sample heated for 90 min (Fig. 4b). The rate of decrease in fluorescence from the Q-TAT SRY and Amel-X/Y amplicons in samples subjected to heating is shown in Fig. 5 and reflects the change in the DNA quantity estimates for each sample using the LMW or HMW standard curves. The intact, male genomic DNA was present in the sample at 200 ng/lL prior to heating. Interestingly, during the first 10 min of heating at 95°C, DNA quantity estimates based upon either Amel-X/Y or SRY appear to increase (Fig. 5). We interpret this phenomenon to reflect a conformational change or “relaxation” of the genomic DNA template that enhances the efficiency with which it is amplified by the Q-TAT multiplex. This initial increase in amplification efficiency was also observed in studies in which degradation was attempted using DNase I (not shown). It is also apparent from Fig. 5 that, whereas the nanogram estimate of DNA in the nonheated sample averages 200 ng/lL using Amel-X/Y, the estimate made using SRY is less, averaging 125 ng/lL. However, at 10 min of heating, there is no statistical difference in DNA quantity estimates made using Amel-X/Y or SRY. The quantity estimates for the sample based upon the AmelX/Y amplicons decrease with heating at a faster rate than the quantity estimates based upon the SRY amplicon as evidenced by the steeper negative slope (Fig. 5). These results support the

Heat (min)

mt287

% of Time 0

mt97

% of Time 0

0 60 90

64,596 5102 986

100.00 7.90 1.53

47676 31,490 20,673

100.00 66.05 43.36

notion that the Q-TAT assay is a useful tool for assessing degradation in a sample. Statistical analysis of 11 repetitions of the degradation experiment summarized in Table 1 and Fig. 5 using one-way ANOVA indicated that after 60 min of heating at 95°C, there is a statistically significant difference in the DNA quantity estimates made using SRY versus the DNA quantity estimates made using Amel-X/Y (Fig. 6). It is also apparent from Table 1 that degradation of mtDNA is revealed with the Q-TAT assay inasmuch as RFU associated with the mt287 amplicon compared with mt97 shows an even more pronounced decline with heating at 95°C. The more evident drop may be explained by the higher molecular weight of the mt287 amplicon relative to Amel-X/Y. To investigate the correlation of Q-TAT results with actual STR genotypes produced from degraded samples, the D3S1358 and D16S539 loci in the Identifiler kit were chosen as reasonable examples of LMW STR alleles and HMW STR alleles, respectively (Life Technologies Inc.). We chose the D16S539 locus because of its similarity in size to the Amel-X and AmelY amplicons in the Q-TAT assay and because fluorescence associated with D16S539 alleles never disappeared entirely during the 90-min heating period. Thus, D16S539 allele-associated fluorescence was always present for quantitative analysis. In contrast, in some cases, fluorescence associated with higher molecular weight STR alleles (e.g., from CSF1P0 and Penta D loci) disappeared completely during the 90-min heating period (Fig. 4b). It appears then that if degradation were detectable for D16S539 alleles, the degradation associated with higher molecular weight STR loci would be even more pronounced (Table 1, Fig. 4b). An evaluation of the STR typing results from a cohort of 37 male reference samples taken from casework that had been previously quantified using Q-TAT and typed using the Identifiler multiplex was performed. Peak height results for the STR alleles (expressed as a ratio of RFU associated with LMW versus HMW alleles) were correlated with the LMW/HMW DNA quantity estimates produced from Q-TAT (Fig. 7). Statistical analysis of results with ANOVA demonstrated a strong correlation between the Q-TAT predictions and actual STR typing results (p < 0.001). Discussion Standard 9.4 of the FBI’s Quality Assurance Standards (14) mandate DNA quantitation of forensic samples using a humanspecific method. The rationale behind the standard is a basic concept of quality assurance with the specific intention of guiding a forensic laboratory to produce an optimal DNA profile on

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Fig. 4––(a and b) Electropherogram of Q-TAT or PowerPlex 16 Hot Start (PP16HS) loci in the DNA samples heated for 0, 60, and 90 min. Standardized DNA was subjected to the heating regimen described in Materials and Methods. Time 0-, 60-, and 90-min samples (the ones described in Table 1) were amplified with either Q-TAT or PP16HS. Products were separated and quantified on a genetic analyzer, and the numbers under each allelic designation represent the peak height.

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(b)

Fig. 4b––Continued

the first attempt; a result likely to occur if the DNA quantity is known. The current widely used technology in place for this purpose is quantitative PCR, and kits are manufactured to quantify total human DNA and male DNA across a broad dynamic range (15,16). The Q-TAT multiplex kit grew out of a response to this quantitation standard, but the strategy behind the assay was for it to be effective and to be compatible with existing laboratory instrumentation. The evolution of the assay has culminated to date with a simple recipe to amplify a DNA extract and to quantify total human and male-specific DNA in a sample (6,7). The assay has been shown to be compatible with robotic liquid-handling

platforms (7), and currently requires about 2 hours to complete (7–13). In addition to detecting and quantifying DNA from a male using the SRY amplicon, additional primers for a 99 bp marker on the X chromosome derived from the hypoxanthine phosphoribosyltransferase gene (HPX) have been incorporated into the Q-TAT assay to provide another marker for quantifying nDNA obtained from a female donor (not shown). To investigate the potential for Q-TAT to indicate the presence of DNA degradation, we performed this study using male DNA quantity estimates produced from standard curves of fluorescence associated with SRY or Amel-X/Y. It is also possible to produce a quantity estimate for total female DNA using Amel-X or Hp (7 and

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Fig. 5––Decay of LMW and HMW amplicons in Q-TAT products in DNA samples heated at 95°C. Genomic DNA from the reference male was heated at 95°C as described. DNA quantity estimates in each sample were made using either the Amel-X+Y (HMW) or SRY (LMW) amplicons and were plotted as a function of heating time. A best fit line was drawn for each data set.

Fig. 6––Ratio of quantity estimates from LMW versus HMW Q-TAT amplicons. Ratios were calculated using LMW amplicon estimate versus HMW amplicon estimate. Deviation of the ratio from that observed for the time 0 sample were subjected to statistical analysis using ANOVA as described using eleven replicates. Bars in the graph bearing asterisks were found to be significant (**)(p < 0.05) to highly significant (***) (p < 0.001) from the 60min time point onward.

unpublished observations). Thus, in a single source female sample, it could be possible to assess degradation in nDNA and mtDNA as well. Published observations demonstrate that successful amplification of a high molecular weight amplicon decreases more rapidly than the successful amplification of a low molecular weight amplicon as DNA becomes increasingly degraded (1–5,16,18). Forensic DNA laboratories at one time or another have seen STR profiles showing clear evidence of degraded genomic DNA template. Often, these profiles will exhibit a sloping of

Fig. 7––Graphical expression of the correlation of LMW/HMW ratios observed for Identifiler amplicons (from the D3S1358 and D16S539 loci) and Q-TAT amplicons (SRY and Amel-X/Y). LMW/HMW DNA quantity estimates (from Q-TAT results) and RFU contained with LMW and HMW STR alleles from the D3 and D16 loci were correlated statistically and found to be significant. Points in each group were connected by lines to better represent the trends.

fluorescence in the peak heights in alleles moving from the low molecular weight to the high molecular weight region of an electropherogram. In some cases, higher molecular weight loci will drop out altogether (19,20). In response to knowing that a sample is degraded, a laboratory can chose to amplify that sample with a more appropriate STR kit, such as a mini-STR kit so that the first attempt at amplification will produce the more complete typing result with that sample (21,22). Thus, in these cases, knowing DNA sample quality or integrity is just as important to successful analysis as knowing DNA sample quantity. The Q-TAT assay produces amplicons in proportion to both the DNA template quantity and DNA template quality by producing a multiplex of amplicons of differing template size (7). In this study, the ability of the assay to provide useful information concerning the state of DNA integrity in an extract from a male donor was demonstrated. Q-TAT results from a standardized DNA sample from a male subjected to controlled degradation demonstrated the assay to be of predictive value in assessing degradation in the DNA template. Moreover, this predictive capability was observed between the Q-TAT results and STR typing results produced from actual casework samples, which underscores the utility of the assay to predict the quality of STR data that will be obtained with a given DNA sample. We chose the D16S539 locus for the HMW alleles because of its similarity in size to the Amel-X and Amel-Y amplicons in the Q-TAT assay. We believed the appearance of degradation within the D16 alleles would be even more pronounced at other high molecular weight STR loci, and this observation was confirmed (Fig. 4b). This was also observed in ratios of HMW/ LMW mtDNA amplicons (the HMW amplicon is almost

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300 bp), with an even more pronounced decrease in the HMW/ LMW ratio in heated DNA samples (Table 1). Moreover, with some nDNA samples heated for 60–90 min, CSF1P0 and Penta D alleles either dropped out completely or one allele fell below threshold making use of D16S539 alleles more reliable for our study (Fig. 4b). Based upon our results, it appears that a quantity estimate ratio of 1.6 or less (LMW/HMW) is characteristic of nDNA capable of producing a full STR profile from a sample. However, above a Q-TAT quantity estimate ratio of about 1.6, the appearance of degradation of DNA template (as evidenced by sloping RFU values from small to large alleles) is increasingly apparent in STR data, and at ratios above 2.5, HMW alleles in the STR profile may begin to drop out of the profile altogether. Any laboratory choosing to use the Q-TAT multiplex to assess degradation would have to identify a threshold ratio below which they would reliably know to opt for a DNA typing analysis method more compatible with partially degraded DNA template, including the use of modified STR kits (such as the Minifiler kit from Life Technologies Inc.), perhaps a panel of SNP markers, or even mtDNA sequencing. Other investigators have explored degradation of DNA in forensic samples (1–5,16–19). In most cases, the rationale applied here to assess degradation was also the rationale for these other approaches. For example, Hudlow et al. (2) developed a qPCR assay that assessed degradation in much the same way as Q-TAT by examining RFU exhibited by HMW versus LMW amplicons amplified in the multiplex qPCR reaction. As expected, qPCR products from large template decreased faster in degraded DNA than did product from small template; however, Hughes-Stamm et al. (1) found no statistically significant correlation between the degradation predicted by the assay and actual sample quality. This result is in contrast to our findings with Q-TAT, which did show a strong correlation between predicted and actual template integrity as assessed by STR typing. Perhaps, our HMW amplicons were larger than those used in the Hughes-Stamm et al. (1) study. Neiderstatter et al. (4) also described qPCR methods that were acceptable for quantifying both nDNA and mtDNA as well as providing an estimate of DNA quality using amplicon quantities from nDNA and mtDNA templates of differing size. Their approach utilized a modular concept involving seven different primer “modules” to quantify nDNA and mtDNA, detect PCR inhibition, and assess DNA degradation (4). In one version of the Q-TAT assay, primers for both nDNA and mtDNA are included in the single multiplex reaction such that both types of DNA can be quantified and characterized for integrity simultaneously. However, quantity estimates for mtDNA are provided in the form of cell equivalents. Simply defined, one cell equivalent (corresponding to 6 pg of nDNA) is assumed to represent about 500 copies of mtDNA. If one knows the number of cell equivalents of mtDNA needed for sequencing, Q-TAT will quantify both nDNA and mtDNA in a sample simultaneously. The authors acknowledge, however, that perhaps further refinement of the assay to extend the dynamic range for mtDNA quantitation is needed. The current assay exhibits linearity in quantifying mtDNA only up to about 250 pg of nDNA (~40 cell equivalents of nDNA and a total of ~20,000 copies of mtDNA, assuming 500 copies of mtDNA/white cell). In addition to quantifying mtDNA, the Q-TAT multiplex is also able to provide an assessment of mtDNA integrity using the same rationale for mtDNA that is used for nDNA (13). In fact, as mt287 is the largest Q-TAT amplicon and mt97 is the smallest, the mt287/mt97 ratio decreases in heat-degraded

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genomic DNA even faster than the ratio of SRY/Amel-X/Y (13, Table 1). One advantage of the Q-TAT approach over other quantitation/characterization methodologies is that the assay provides a complete picture of the DNA sample under investigation. Information concerning template DNA quantity (both total and male, nDNA and mtDNA) and template DNA integrity are routinely returned to the investigator from the results as is whether or not PCR inhibitors exist in the extract (7). The utility of an assay-like Q-TAT for a forensic laboratory that works with evidentiary samples that may exist in elevated states of decomposition cannot be overstated. The magnitude of the difficulty of producing STR results from degraded samples was made crystal clear identifying human remains from the 2001 World Trade Center attack (23). Laboratories that specialize in identifying human remains would therefore benefit greatly from routine use of an assay capable of accurately assessing the state of degradation of DNA recovered from remains (23,24). With the information made available to the DNA analyst from an assay-like Q-TAT, choices regarding how best to proceed with DNA analysis on the sample can be made. If the quality of the DNA is so bad as to predict that only limited STR information will be forthcoming, an analyst might choose to begin testing with one of the mini-STR kits available or perhaps opt for testing with some of the SNP panels that are becoming available as well (17–19,21,22). References 1. Hughes-Stamm SR, Ashton JJ, van Daal A. Assessment of DNA degradation and the genotyping success of highly degraded samples. Int J Legal Med 2011;125:341–8. 2. Hudlow WR, Chong MD, Swango KL, Timken MD, Buoncristiani MR. A quadruplex real-time qPCR assay for the simultaneous assessment of total human DNA, human male DNA, DNA degradation and the presence of PCR inhibitors in forensic samples: a diagnostic tool for STR typing. Forensic Sci Int Genet 2008;2:108–25. 3. Swango KL, Timken MD, Chong MD, Buoncristiani MR. A quantitative PCR assay for the assessment of DNA degradation in forensic samples. Forensic Sci Int 2006;158:14–26. 4. Niederstatter H, Kochl S, Grubwieser P, Pavlic M, Steinlechner M, Parson W. A modular real-time PCR concept for determining the quantity and quality of human nuclear and mitochondrial DNA. Forensic Sci Int Genet 2007;1:29–34. 5. Nicklas JA, Noreault-Conti T, Buel E. Development of a real-time method to detect DNA degradation in forensic samples. J Forensic Sci 2012;57:466–71. 6. Allen RW, Fuller VM. Quantitation of human genomic DNA through amplification of the amelogenin locus. J Forensic Sci 2006;51:76–81. 7. Wilson JP, Fuller VM, Benson G, Juroski D, Duvall E, Fu J, et al. Molecular assay for screening and quantitating DNA in biological evidence: the modified Q-TAT assay. J Forensic Sci 2010;55:1050–7. 8. Henry FM, Smith BC, Wilson JP, Fuller VM. Evaluating a means of reducing the current cost of setting up a DNA lab through equipment choice and a novel multiplexing and gender-typing quantification method. Proceedings of the 21st International Symposium on Human Identification; 2010 Oct 11–14; San Antonio, TX. Madison, WI: Promega Corporation, 2010. 9. Smith BC. Evaluating DNA sample degradation with a quantitative gender typing end-point PCR multiplex [Master’s thesis]. Stillwater, OK: Oklahoma State University, 2011 Dec. 10. Fuller VM. Accessibility and the backlog: rethinking DNA capability and capacity. Forensic Mag 2011; April-May:15–9. 11. Shewale J, Richey SL, Sinha SK. Anomalous amplification of the amelogenin locus typed by AmpFLSTR Profiler Plus amplification kit. Forensic Science Communications 2000; www.fbi.gov/hq/backissu/oct2000/ shewale. 12. Berta P, Hawkins JR, Sinclair AH, Taylor A, Griffiths BL, Goodfellow PN, et al. Genetic evidence equating SRY and the testes determining factor. Nature 1990;348:448–50.

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13. Vandegrift EA. Quantitation and characterization of human nuclear and mitochondrial DNA with PCR and capillary electrophoresis [master’s thesis]. Stillwater (OK): Oklahoma State University, 2010. 14. http://www.fbi.gov/about-us/lab/biometric-analysis/codis/qas-standardsfor-forensic-dna-testing-laboratories-effective-9-1-2011. 15. Heid CA, Stevens J, Livak JJ, Williams PM. Real-time quantitative PCR. Genome Res 1996;6:986–94. 16. https://tools.invitrogen.com/content/sfs/manuals/cms_041395.pdf 17. Fondevila M, Phillips C, Navera0 n N, Cerezo M, Rodrıguez A, Calvo R, et al. Challenging DNA: assessment of a range of genotyping approaches for highly degraded forensic samples. Forensic Sci Int Genet 2008;1 (Suppl):26–8. 18. Sanchez JJ, Børsting C, Balogh K, Berger B, Bogus M, Butler JM, et al. Forensic typing of autosomal SNPs with a 29 SNP-multiplex— results of a collaborative EDNAP exercise. Forensic Sci Int Genet 2008;2:176–83. 19. Dixon LA, Dobbins AE, Pulker HK, Butler JM, Vallone PM, Coble MD, et al. Analysis of artificially degraded DNA using STRs and SNPs —results of a collaborative European (EDNAP) exercise. Forensic Sci Int 2006;164:33–44. 20. Butler JM. Forensic DNA typing: biology, technology, and genetics of STR markers, 2nd ed. New York, NY: Elsevier, 2005. 21. Grubweiser P, Muhlmann R, Berger B, Niederstatter H, Pavlic M, Parson W. A new “miniSTR-multiplex” displaying reduced amplicon

22. 23. 24. 25.

lengths for the analysis of degraded DNA. Int J Legal Med 2006;120:115–20. Hill CR, Kline MC, Coble MD, Butler JM. Characterization of 26 miniSTR loci for improved analysis of degraded DNA samples. J Forensic Sci 2008;53:73–80. Mundorff AZ, Bartelink EJ, Mar-Cash E. DNA preservation in skeletal elements from the world trade center disaster: recommendations for mass fatality management. J Forensic Sci 2009;54:739–45. Lee HY, Kim NY, Park MJ, Sim JE, Yang WI, Shin KJ. DNA typing for the identification of old skeletal remains from Korean war victims. J Forensic Sci 2010;55:1422–9. Allen RW, Fuller VM, inventors. Oklahoma State University, assignee. Method for simultaneously determining in a single multiplex reaction gender of donors and quantities of genomic DNA and ratios thereof, presence and extent of DNA degradation, and PCR inhibition within a human DNA sample. US patent 8,153,372, B2. April 10.

Additional information and reprint requests: Robert W. Allen, Ph.D. School of Forensic Sciences Center for Health Sciences 1111 West 17th Street Tulsa, OK 74107 E-mail: [email protected]

Evaluation of degradation in DNA from males with a quantitative gender typing, endpoint PCR multiplex.

Evidentiary samples submitted to a forensic DNA laboratory occasionally yield DNA that is degraded. Samples of intact chromosomal DNA (both nuclear an...
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