The effect of an enzymatic bone processing method on short tandem repeat profiling of challenged bone specimens Richard Li ⇑ , Stacey Klempner Forensic Science Program, Department of Science, John Jay College of Criminal Justice, The City University of New York, New York, NY 10019, USA article info Article history: Received 18 August 2012 Received in revised form 21 December 2012 Accepted 26 December 2012 Available online 4 February 2013 Keywords: Bone Trypsin Forensic DNA STR abstract Forensic analysis of DNA from bone can be important in investigating a variety of cases involving violent crimes and mass fatality cases. To remove the potential presence of co-mingled remains and to eliminate contaminants that interfere with forensic DNA analysis, the outer surface of the bone fragment must be cleaned. This study evaluated two methods for processing bone specimens prior to DNA isolation. Mechanical sanding and enzymatic trypsin methods were compared in this study. The effects of these methods on the yield of DNA isolated and the quality of DNA analysis were studied. It was revealed that comparable values of DNA yields between the two methods were observed. Additionally, to evaluate the capabilities of the cleaning effect of the bone processing methods, the presence of polymerase chain reac- tion inhibitors in the DNA extracts was monitored using the internal positive control. Similar C t values of the internal positive control were observed as the DNA extracts of the trypsin method compared with that of the sanding method. The characterization of the effects of the trypsin treatment on the quality of DNA profiling was also carried out. To evaluate the integrity of the nuclear DNA isolated, the percent- age of allele calls and the peak-height values of alleles of the short tandem repeat profiles were compared between the two methods. A paired-sample t-test revealed no significant difference between the two methods. Our data suggested that the trypsin method can be used as an alternative cleaning method to mechanical cleaning methods. This method can be used to process multiple samples simultaneously. This can be very important for achieving high-throughput DNA isolation through potential automation, which can be extremely valuable for situations such as the forensic DNA analysis of skeletal remains from mass fatality incidents. Ó 2012 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Forensic analysis of DNA from bone can be important in inves- tigating a variety of cases involving violent crimes and mass fatal- ity cases [1–5]. To assure the accuracy of the forensic DNA analysis, bone samples must be appropriately processed prior to DNA isola- tion. Bone samples collected from crime scenes have potential con- tamination by presence of co-mingled remains and by physical contact of emergency dispatch personnel [6–8]. Additionally, bur- ied bones usually contain polymerase chain reaction (PCR) inhibi- tors that interfere with forensic DNA analysis [9,10]. Thus, an outer surface layer (approximately 1–2 mm) of bone fragment should be removed. This is usually carried out using mechanical methods, such as sanding using sanding discs attached to a rotary tool [3,11,12] or sandpaper [13,14]. In our previous study, an enzymatic method using trypsin solu- tion [15,16] was adapted to the sample cleaning method prior to DNA isolation from fresh bone samples [17,18]. Microscopic stud- ies demonstrated that this trypsin method is effective for the re- moval of outer surface materials such as the mineralized bone connective tissue of fresh human bone samples. The yield of DNA isolated from trypsin-treated fresh bone samples was sufficient for subsequent short tandem repeat (STR) analysis. In this study, the application of the enzymatic trypsin method for DNA isolation was studied in samples that are more typically encountered in ac- tual forensic cases. Additionally, the yield and the quality of DNA extracted from challenged bones were compared between the mechanical sanding and enzymatic trypsin method side-by-side. 2. Materials and methods 2.1. Sample preparation Precautions were followed to eliminate possible DNA contami- nants. Sampling was carried out in a sterilized laminar flow cabi- net. Consumables were DNA free. Disposable laboratory coats, gloves, and masks were used throughout the procedure. 1344-6223/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.legalmed.2012.12.002 ⇑ Corresponding author. Tel.: +1 646 557 4886. E-mail address: [email protected](R. Li). Legal Medicine 15 (2013) 171–176 Contents lists available at SciVerse ScienceDirect Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Transcript
Legal Medicine 15 (2013) 171–176
Contents lists available at SciVerse ScienceDirect
The effect of an enzymatic bone processing method on short tandem repeatprofiling of challenged bone specimens
Richard Li ⇑, Stacey KlempnerForensic Science Program, Department of Science, John Jay College of Criminal Justice, The City University of New York, New York, NY 10019, USA
a r t i c l e i n f o a b s t r a c t
Article history:Received 18 August 2012Received in revised form 21 December 2012Accepted 26 December 2012Available online 4 February 2013
Keywords:BoneTrypsinForensicDNASTR
1344-6223/$ - see front matter � 2012 Elsevier Irelanhttp://dx.doi.org/10.1016/j.legalmed.2012.12.002
Forensic analysis of DNA from bone can be important in investigating a variety of cases involving violentcrimes and mass fatality cases. To remove the potential presence of co-mingled remains and to eliminatecontaminants that interfere with forensic DNA analysis, the outer surface of the bone fragment must becleaned. This study evaluated two methods for processing bone specimens prior to DNA isolation.Mechanical sanding and enzymatic trypsin methods were compared in this study. The effects of thesemethods on the yield of DNA isolated and the quality of DNA analysis were studied. It was revealed thatcomparable values of DNA yields between the two methods were observed. Additionally, to evaluate thecapabilities of the cleaning effect of the bone processing methods, the presence of polymerase chain reac-tion inhibitors in the DNA extracts was monitored using the internal positive control. Similar Ct values ofthe internal positive control were observed as the DNA extracts of the trypsin method compared withthat of the sanding method. The characterization of the effects of the trypsin treatment on the qualityof DNA profiling was also carried out. To evaluate the integrity of the nuclear DNA isolated, the percent-age of allele calls and the peak-height values of alleles of the short tandem repeat profiles were comparedbetween the two methods. A paired-sample t-test revealed no significant difference between the twomethods. Our data suggested that the trypsin method can be used as an alternative cleaning methodto mechanical cleaning methods. This method can be used to process multiple samples simultaneously.This can be very important for achieving high-throughput DNA isolation through potential automation,which can be extremely valuable for situations such as the forensic DNA analysis of skeletal remains frommass fatality incidents.
� 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Forensic analysis of DNA from bone can be important in inves-tigating a variety of cases involving violent crimes and mass fatal-ity cases [1–5]. To assure the accuracy of the forensic DNA analysis,bone samples must be appropriately processed prior to DNA isola-tion. Bone samples collected from crime scenes have potential con-tamination by presence of co-mingled remains and by physicalcontact of emergency dispatch personnel [6–8]. Additionally, bur-ied bones usually contain polymerase chain reaction (PCR) inhibi-tors that interfere with forensic DNA analysis [9,10]. Thus, an outersurface layer (approximately 1–2 mm) of bone fragment should beremoved. This is usually carried out using mechanical methods,such as sanding using sanding discs attached to a rotary tool[3,11,12] or sandpaper [13,14].
In our previous study, an enzymatic method using trypsin solu-tion [15,16] was adapted to the sample cleaning method prior to
d Ltd. All rights reserved.
DNA isolation from fresh bone samples [17,18]. Microscopic stud-ies demonstrated that this trypsin method is effective for the re-moval of outer surface materials such as the mineralized boneconnective tissue of fresh human bone samples. The yield of DNAisolated from trypsin-treated fresh bone samples was sufficientfor subsequent short tandem repeat (STR) analysis. In this study,the application of the enzymatic trypsin method for DNA isolationwas studied in samples that are more typically encountered in ac-tual forensic cases. Additionally, the yield and the quality of DNAextracted from challenged bones were compared between themechanical sanding and enzymatic trypsin method side-by-side.
2. Materials and methods
2.1. Sample preparation
Precautions were followed to eliminate possible DNA contami-nants. Sampling was carried out in a sterilized laminar flow cabi-net. Consumables were DNA free. Disposable laboratory coats,gloves, and masks were used throughout the procedure.
172 R. Li, S. Klempner / Legal Medicine 15 (2013) 171–176
2.1.1. SamplingIn this study, challenged human bone specimens (non-proba-
tive) were used (Fig. 1). A total of 14 bone specimens (a similarsample size as in Loreille et al. [12]) including cranium, rib, and ti-bia from different individuals were selected. Aged bones includingburied bones unearthed from archaeological sites were included.Bones ranging in age from approximately 50 to over 100 years
Fig. 1. Typical bone fragment specimens investigated in this study. (A) Rib, buriedfor approximately 50 years with possible maceration using bleach after unearthing,stored at room temperature; (B) parietal bone, autopsy specimen, possiblymacerated using boiling, stored at room temperature for approximately 65 years;and (C) tibia (no DNA detected), buried for over 100 years under high heat andhumidity, stored at room temperature.
post-mortem were selected for this study. Bones exposed to poten-tial insults (such as the possibility of maceration using bleaching orboiling, or buried under high heat and humidity) were included.
A pair of bone fragments (approximately 1 g each) was dis-sected from each bone specimen. A pair of bone fragments wasthen processed using the sanding and trypsin method separatelyfor pair-wise comparisons. A paired-sample t-test (two-tail) wasconducted to compare the data from the sanding and trypsin meth-ods in this study.
2.1.2. Surface cleaningThe trypsin treatment was carried out as developed previously
[17]. It was initiated by adding 5 ml of fresh trypsin (Fisher Scien-tific) solution (30 lg/ll, 10 mM Tris, pH 7.5) to the bone fragmentand then was incubated at 55 �C with gentle agitation for 2 h. Afterincubation, the supernatant was removed. The sanding was carriedout using a current sanding method [11] with single-use sandingdiscs attached to a rotary sanding tool (Dremel, Racine, WI). Theouter surfaces were sanded thoroughly. The cleaned bone frag-ments using both methods were further processed by inversionfor 30 s in distilled water, 0.5% sodium hypochloride, and 96% eth-anol as described in Davoren et al. [11]. The bone fragments werethen air dried.
2.1.3. Bone tissue disruptionThe pulverized bone powder from each fragment was prepared
using the freezer mill method [12] that utilizes a cryogenic impactgrinder (SamplePrep 6770 Freezer Mill, SPEX, Metuchen, NJ). Theprocedure was programmed according to the manufacturer’s pro-tocols: 10 min pre-cooling followed by 2 cycles of grinding(2 min grinding at a rate of 20 impacts/s and 2 min cooling for eachcycle).
2.2. DNA extraction
Demineralization of pulverized bone powder was carried out asdescribed in Loreille et al. [12]. For each sample, 0.2 g of pulverizedbone powder was decalcified by incubating in 3.2 ml of extractionbuffer (0.5 M EDTA, 1% lauryl-sarcosinate) and 200 ll of 20 mg/mlproteinase K overnight at 56 �C with gentle agitation.
Table 1Summary of pair-wise comparisons of DNA quantitation results.
a Buried bones (buried for approximately 50 years) with possible macerationusing bleach after unearthing were stored at room temperature.
b Autopsy specimens, possible maceration using boiling, were stored at roomtemperature for approximately 65 years.
Fig. 2. The effect of trypsin treatment on STR profiles. STR profiles were obtained with the AmpF‘STR MiniFiler amplification kit. The electropherograms of (A) sanded and(B) trypsin-treated sample (JJC1516).
R. Li, S. Klempner / Legal Medicine 15 (2013) 171–176 173
The DNA from each sample was extracted using the methodadapted from Courts and Madea [19] with slight modifications.The volume of the demineralized sample was reduced to approxi-
mately 400 ll using an Amicon Ultra-4 (30 kDa) column (Millipore,Billerica, MA). The small molecular weight cutoffs of the centrifu-gal device retain a greater number of short DNA elements, thereby
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increasing the potential for successful DNA amplification [20]. DNAwas extracted using the QIAquick PCR Purification Kit (QIAGEN,Valencia, CA) according to the manufacturer’s protocols. The finalvolume of eluted DNA was 60 ll. Extraction negative controls wereemployed to monitor the potential contaminations. DNA profilesfrom laboratory personnel were available for the detection of lab-oratory DNA contaminations.
2.3. DNA quantification of nuclear DNA using real time PCR
DNA quantitation was performed using the Quantifiler HumanDNA Quantitation kit (Applied Biosystems, Foster City, CA) on anABI 7300 Real Time PCR System (Applied Biosystems) accordingto the manufacturer’s protocols. Internal positive controls (IPC)were used to monitor PCR inhibitors present in the DNA extracts.Negative controls and reagent blanks were included to monitor po-tential contamination. DNA yield was expressed as ng DNA/g bone.
2.4. STR analysis
STR analysis was performed using the AmpF‘STR MiniFileramplification kit (Applied Biosystems). For each amplification,0.5 ng of DNA sample was used according to the manufacturer’sinstructions. A positive control with genomic DNA of a knownSTR profile was included. Extraction and PCR negative controlswere also amplified to monitor potential contamination. Amplifiedproducts were separated on an Applied Biosystems 310 GeneticAnalyzer, and results were analyzed with GeneMapper ID software.A 50 relative fluorescent units (rfu) threshold for allele designationwas used, thus peaks below 50 rfu were disregarded.
0
20
40
60
80
100
Am
plifi
catio
n Su
cces
s (%
of S
TR a
llele
cal
ls)
STR Loci
Sanding Trypsin
Fig. 3. The amplification success at each locus of the AmpF‘STR MiniFiler (thenumber of allele calls compared to the highest number of alleles detected).
3. Results
3.1. The characterization of the effects of trypsin treatment on the yieldof DNA isolated
To characterize the effect of trypsin treatment on the yield andthe quality of DNA isolated, DNA extracts were compared side-by-side: (1) DNA extracts isolated from the sanding method, and (2)DNA extracts isolated with the trypsin method.
To find out if this trypsin method achieved sufficient DNA yieldfor forensic DNA profiling, total DNA was isolated and quantified.Among 14 bone specimens, DNA was detected from 9 bone speci-mens. The negative quantitation results from 5 buried bones weremost probably caused by highly degraded template DNA. Thus, theDNA extracts derived from those 9 bone specimens were studiedfurther. The DNA yield from different bone specimens, ranged from2.25 to 7341 ng DNA/g bone, was highly variable (Table 1). TheDNA yields of trypsin-treated samples were slightly higher thanthat of sanded samples. The average ratio of DNA yield isolatedfrom trypsin treated versus sanded bone samples was 1.66 (witha standard deviation of the average ratio at 1.87). A paired-samplet-test (two-tail) was conducted to compare the yields of the sand-ing and trypsin methods. There was no significant difference in theyields of the two methods (p value = 0.351).
PCR inhibitors cause inhibitions in PCR-based forensic DNAanalysis [21]. To evaluate the capabilities of the cleaning effect ofthe bone processing method, the presence of PCR inhibitors inthe DNA extracts was measured by monitoring the amplificationof the IPC. The Quantifiler Human DNA Quantitation kit (see Sec-tion 2) contains a known amount of exogenous DNA as IPC thatcan be fortified to the sample and amplified. Monitoring the ampli-fication of IPC enables the detection of PCR failure due to inhibitionwhen the IPC Ct value is relatively higher than that of an uninhib-ited PCR reaction. Our results revealed that similar IPC Ct values
were observed as the DNA extracts of the trypsin method werecompared with that of the sanding method. The average IPC Ct val-ues were 28.49 for the sanding method and 28.24 for the trypsinmethod, respectively. The average ratio of IPC Ct value from trypsintreated versus sanded bone samples was 0.99 (with a standarddeviation of the average ratio at 0.011). A paired-sample t-test(two-tail, comparing the sanding and trypsin methods) indicatedthat there was no significant difference in the IPC Ct values of thetwo methods (p value = 0.596). No PCR inhibiting substances weredetected.
3.2. The characterization of the effects of trypsin treatment on thequality of STR analysis
To find out if this processing method caused any adverse effecton DNA, the quality of DNA profiles was evaluated. DNA sampleswere examined using STR analysis. In highly degraded specimens,however, STR analysis using standard multiplex STR kits may havedifficulties in amplifying larger sized alleles [19,20]. As the high de-gree of DNA template fragmentation occurs in degraded speci-mens, the abundance of complete target fragments is greatlyreduced. As a result, partial STR profiles are often observed undersuch condition. Since the bone specimens used in this study wereexposed to acidic soils and potential insults such as bleach andboiling, this may lead to DNA template degradation [20]. To in-crease success in STR analysis, we utilized the AmpF‘STR MiniFileramplification kit (Applied Biosystems) with primer pairs that pro-duce shorter amplicons than that of a standard STR kit such as theAmpF‘STR Identifiler PCR Amplification Kit. Thus, it is usually moresuccessfully performed than standard STR kits for degraded DNAsamples [22]. STR analysis was carried out only on samples show-ing detectable DNA in the quantitative PCR assay. A total of 18 DNAsamples (9 pairs of sanded versus trypsin-treated samples) derivedfrom 9 bone specimens were tested.
To evaluate the integrity of the DNA isolated, the percentage ofallele calls was compared. Only peaks with a peak height of 50 rfuor more were included. Profile results were compared betweensanding and trypsin-treated samples side-by-side, allowing foreasy detection of allelic drop-out. Successfully typed loci were ob-served in both sanded and trypsin-treated samples. Some samples(4 pairs of samples), particularly for larger size alleles at D7S820,D18S51, D21S11, and FGA loci, exhibited drop-outs, probably due
Table 2Comparisons of average peak heights at each locus of the AmpF‘STR MiniFiler in rfu.
R. Li, S. Klempner / Legal Medicine 15 (2013) 171–176 175
to template degradation. An example of such STR profile is shownin Fig. 2. Overall, the percentage of allele calls of the trypsin meth-od was similar as to that of the sanding method (Fig. 3). A paired-sample t-test (two-tail) revealed no significant difference betweenthe two methods (p value = 0.681).
The integrity of the DNA samples was also examined by com-paring the average rfu values of each allele at each locus betweensanded and trypsin-treated samples. Only alleles showing success-ful amplification in both sanded and trypsin-treated samples werecompared. The rfu values of the trypsin-treated samples were com-parable to those of the sanded samples (Table 2). A paired-samplet-test (two-tail) revealed that no significant difference in peakheights was detected in rfu between two methods (p value > 0.05).
4. Discussion
In this study, the feasibility of using the enzymatic trypsinmethod for cleaning bones prior to DNA isolation was examined.It was revealed that comparable values of DNA yields betweenthe mechanical sanding and enzymatic trypsin method were ob-served. Additionally, to evaluate the capabilities of the cleaning ef-fect of the methods, the presence of PCR inhibitors in the DNAextracts was monitored using IPC. Similar IPC Ct values were ob-served as the DNA extracts of the trypsin method were comparedwith that of the sanding method. The characterization of the effectsof the trypsin method on the quality of DNA profiling was also car-ried out. In the evaluation of the integrity of the genomic DNA iso-lated, the percentage of the allele calls of STR profiles and the rfuvalues of STR alleles were comparable between the two methods.A paired-sample t-test revealed no significant difference betweenthe two methods. The results suggested that the effects of the tryp-sin method were comparable with the sanding method.
However, mechanical methods have some disadvantages. Thesemethods cannot easily be adapted for automation. Thus, it cannotbe used to process multiple samples simultaneously. When thebone surface is porous or fragile, surface cleaning using a mechan-ical method is not possible [23]. Moreover, the bone dust generatedby mechanical methods is a potential source of cross-contamina-tion between samples [24]. This study suggests that the trypsinmethod can be used as an alternative to mechanical methods forhuman bone samples. This powder-less cleaning method can beused to process multiple samples simultaneously. This can be veryimportant for achieving high-throughput DNA isolation throughpotential automation, which can be extremely valuable for situa-tions such as the forensic DNA analysis of skeletal remains frommass fatality incidents. Additionally, this cleaning method mayhave a low risk of cross contamination between samples from air-borne bone powder dust. Additionally, it provides a safe method ofbone cleaning to prevent exposure to bone powder dust, whichmay carry blood–borne pathogens.
Acknowledgments
This study was supported by grants 2009-DN-BX-K209,awarded to R.L. by the National Institute of Justice from the USDepartment of Justice. Points of view in this publication are thoseof the authors and do not necessarily represent the official positionor policies of the US Department of Justice. We thank the AmericanMuseum of Natural History for providing useful specimens for thisstudy. We also thank Dr. Ian Tattersall, Gisselle Garcia-Pack, Kris-ten Mable, and Zully Santiago for helpful discussions andassistance.
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