Legal Medicine 14 (2012) 272–275

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Legal Medicine

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Brief Communication

DNA extraction method from bones using Maxwell� 16

Monika Karija Vlahovic ⇑, Milovan KubatDNA Laboratory, Department of Forensic Medicine and Criminology, School of Medicne, Šalata 11, HR-10000 Zagreb, Croatia

a r t i c l e i n f o a b s t r a c t

Article history:Received 1 March 2012Received in revised form 12 April 2012Accepted 13 April 2012Available online 23 May 2012

Keywords:BoneExtractionAutomationCroatia

1344-6223/$ - see front matter � 2012 Elsevier Irelanhttp://dx.doi.org/10.1016/j.legalmed.2012.04.004

⇑ Corresponding author. Tel.: +385 1 4590 232; faxE-mail address: mkarija@mef.hr (M. Karija Vlahov

This paper describes the automated purification of DNA extracted from human bones using Maxwell� 16bench top instrument. Analysis of nuclear short tandem repeats (STR) is invaluable in identification ofhuman remains exhumed from mass graves in Croatia. Up to today 4683 skeletal remains have beenrecovered and for 897 human remains identity has not been determined. DNA has been extracted from70% of all unidentified samples. For more than 90% of the samples nuclear STR profiles have beenobtained using either organic phenol/chloroform method or silica-column purification for the extractionof DNA from bones or teeth. In order to evaluate a Maxwell� 16 DNA extraction performance 40 bonesamples with different stage of decomposition were analyzed. The efficacy of manual silica based extrac-tion and an automated purification was compared. The DNA yield per gram of starting material, removalof inhibitors and the quality of resulting STR profiles of the Maxwell extracts from duplicate amplifica-tions were evaluated. The results show that Maxwell 16 platform can be used instead of manual DNAextraction procedures.

� 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Identification of victims from the 1991–1995 war in Croatia isan ongoing process. From 1996 till today human remains of a totalof 4683 individuals have been recovered from 144 mass graves.Three thousand seven hundred and eighty six remains were suc-cessfully identified. Many of them were identified by standardforensic identification methods which includes anthropological,X-rays and dental examination [1,2]. However DNA analysis wasused in cases where premortal records are missing and samplesare badly decomposed [3]. DNA analysis has played a significantrole in more than 1130 identification cases from 41 mass graves.Bone and teeth were/are often the only available samples forDNA analysis. A protocol which uses phenol/chloroform extractionhas been used from the very beginning in our laboratory [4–7].Also a bone extraction method based on the Qiagen Mini Kit’s pro-tocol (QIAamp DNA Mini Kit, QIAGEN, Hilden, Germany) with somemodification was successfully used for the last 4 years [8,9]. Herewe present extraction protocols using silica columns and Maxwell�

16 Forensic Instrument System (Promega Corporation, Madison,WI, USA) [8,10]. DNA was extracted from 40 randomly chosen bonesamples. The performance of extraction method using Maxwell�

16 (Promega) was monitored via Real-time PCR and amplificationof STR loci using AmpFlSTR� Identifiler� Plus PCR AmplificationKit (Applied Biosystems, Foster City, CA, USA).

d Ltd. All rights reserved.

: +385 1 4590 235.ic).

2. Materials and methods

Cleaning and pulverization of skeletal samples, DNA extraction,quantification and amplification setup were conducted following agenerally accepted safety and DNA typing guidelines.

2.1. Samples

Forty samples marked S1–S40 with different histories and de-grees of degradation were used. The samples were as follows:S39 was a 1 year old bone found in a river, S40 was a 1 year oldbone buried in soil, three bones were 65 years old (S2, S3, S29)and the rest of the samples were excavated remains of personskilled between 1991 and 1995. The history of the excavated boneswas as follows: 13 bones were 20 years old, one bone was 19 yearsold, one bone was 17 years old and the rest of the 20 bones were15 years old skeletal remains. We used long bones (femur, hu-merus, tibia, ulna and radius) with the exception of two fragmentsof the cranial bones.

2.2. Sample preparation

The surface of the skeletal element was removed using a rotarysanding machine (Dremel, Racine, WI, USA) in order to eliminatepotential contamination and dirt. The samples were then cut intothin slices and washed twice with 10% sodium hypochlorite for15 min, then twice rinsed and soaked in water for 15 min, oncerinsed in 70% ethanol and finally rinsed in 96% ethanol. Thesamples were left to dry overnight at room temperature. Frozen

M. Karija Vlahovic, M. Kubat / Legal Medicine 14 (2012) 272–275 273

slices were pulverized using MM301 mill (Retsch, Haan, Germany).Every bone was prepared separately using sterile tools in a spe-cially designed room.

2.3. DNA extraction using silica columns

One gram of bone powder was incubated in 10 mL of 0.5 M EDTApH 8.5 solution for 24 h at room temperature. The next day thesupernatant was discarded, and the EDTA solution was changedtwo more times. After 72 h of decalcification the precipitate waswashed twice with sterile water. Decalcified pellets and reagentblank were then extracted using QIAamp Mini Kit (QIAGEN, Hilden,Germany). Bone powders were dissolved in 2 mL of ATL buffer (QIA-amp DNA Mini Kit, QIAGEN, Hilden, Germany) with 2 mg of Protein-ase K (Gibco, Invitrogen Corporation, Carlsbad, CA). After incubationat 56 �C for 24 h, 1.6 mL of AL buffer (QIAamp DNA Mini Kit, QIA-GEN, Hilden, Germany) was added and the mixture was incubatedfurther at 70 �C for 1 h. The tubes were centrifuged for 5 min at�1000g to pellet any particles and clear lysate was transferred toa sterile tube. One volume of 96% EtOH was added and the mixturewas spun through a QIAamp Mini Spin column (QIAGEN, Hilden,Germany). Due to the large volume of the liquid, the samples werespun through the column several times. Columns were centrifugedfor 2 min at 2000g each time the liquid was added. The membraneof the QIAamp Mini column was washed with 700 lL AW1 buffer(QIAamp DNA Mini Kit, QIAGEN, Hilden, Germany) following thecentrifugation at 2000g for 5 min. After adding of 700 lL AW2 buf-fer (QIAamp DNA Mini Kit, QIAGEN, Hilden, Germany) the columnswere centrifuged for 15 min at 2000g. The DNA was eluted in300 lL of previously heated AE buffer. After centrifugation at2000g for 2 min, another 300 lL of AE buffer was added. The final600 lL of elute was applied to Amicon Ultra 100K device (Millipore,Billerica, MA, USA) and concentrated to 50 lL.

2.4. Automated DNA purification using Maxwell� 16

The same set of samples was used to test performance of auto-mated DNA purification with use of DNA IQ™ Casework Pro Kit forMaxwell� 16 (Promega Corporation, Madison, WI, USA). One gramof bone powder was decalcified as previously described. Decalci-fied bone material was incubated in 3 mL of Bone Incubation Buffer(1 M NaCl, 0.1 M Tris pH 8.0, 0.1 M EDTA pH 8.0, 20% SDS) with2 mg of Proteinase K (Invitrogen) at 56 �C over night. The secondday the lysate was centrifuged to pellet any particles and the clearsolution was concentrated to the volume of 300 lL using AmiconUltra 100K device (Millipore). According to the manual [9], forsamples with a starting volume between 250 and 330 lL thevolume of 420 lL of Lysis buffer containing DTT (DNA IQ CaseworkPro Kit, Promega) should be added. Following the manufacturer’srecommendation, 300 lL of concentrated lysate was mixed with420 lL of Lysis Buffer containing DTT (DNA IQ Casework Pro Kit,Promega) and the liquid was transferred to a prefilled reagent car-tridge. Fifteen samples and a reagent blank were processed at thesame time. After 30 min the purification process was completedand DNA was eluted in 50 lL of Elution Buffer (Promega).

2.5. DNA quantification

The DNA extracts from all samples were quantified in duplicateusing the Quantifiler Human DNA Quantification Kit (AppliedBiosystems, Foster City, CA, USA) and 7000 Sequence System(Applied Biosystems) using SDS Software v1.2 according to themanufacturer’s recommendation. Ct values for Internal PositiveControls (IPC) were monitored to evaluate the levels of PCR inhibi-tion. For each isolate DNA yield per gram of bone powder wascalculated.

2.6. STR typing and analysis

All DNA extracts were amplified in duplicate using the Amp-FlSTR� Identifiler � Plus PCR Amplification Kit (Applied Biosys-tems) in order to confirm genetic profile of the bone samples.Prior to the amplification the extracts, where possible, were nor-malized to 0.1 ng/lL. Ten microliters of the extract was used inthe PCR reaction and the 29 PCR cycle protocol was selectedaccording to manufacturer’s recommendations.

In all amplification reactions a positive control and negative PCRcontrols were used, as well as reagent blanks from extractions.

The STR fragments were separated by capillary electrophoresisusing 3100-Avant Genetic Analyzer (Applied Biosystems), POP4polymer and the collection software 3100-Avant Data Collectionv2.0. The samples were genotyped using GenemapperID v3.2.1software.

The genetic profiles obtained for the bone samples were com-pared to the STR profiles of all employees involved in the processto monitor possible contamination.

3. Results and discussion

3.1. DNA quantification

Real-time PCR quantification results for two extraction proce-dures are presented in Table 1. The average amount of DNA recov-ered per gram of starting bone powder using the silica method was6.25 and 7.08 ng/g for Maxwell extracts. DNA concentration wasnot determined for nine femurs and one cranial bone extractedby the manual silica method: samples S4, S5, S8, S10, S11, S12,S14, S15, S24 and S27. For the same set of samples with theMaxwell protocol (except the sample S24), DNA concentration ran-ged between 0.0292 and 0.835 ng/lL (Table 1). We detected lessthan 0.023 ng of DNA/lL in 10 Qiagen extracts and no DNA from10 samples.

The Maxwell extraction method yielded a higher amount ofDNA than the manual purification procedure for 21 samples. Quan-tification of nuclear DNA extracted by the Maxwell 16 platformwas successful for all extracts but one fragment of the skull(sample S25). We detected more than 0.023 ng DNA/lL of isolatefrom 26 bones, less than 0.023 ng DNA/lL of isolate from 13 bonesand no DNA from one sample (Table 1).

Although some samples yielded better results when the Qiagenprocedure was used, obtained quantification results of the Maxwellextracts suggest that robotic platform can be used in cases of de-graded samples as well. Ct IPC values for quantification standardDNA samples were around 26 cycles while extracts obtained fromboth tested methods required on average 26.5 cycles to reach theearly log phase of the PCR reaction. The result indicates that the ex-tracted DNA (manual and robotic) contains low levels of inhibitors.

3.2. STR analysis

The quantity and purity of extracted DNA using the Maxwell 16purification system was further evaluated by STR analysis. All ex-tracts were amplified in duplicate and only the consensus loci fromtwo amplifications were reported. It is important to note that theextracts prior to the PCR setup, when possible, were normalizedto 0.1 ng/lL and 10 lL of the extract was used in each amplifica-tion reaction. The mean peak height (RFU) was examined for bothextraction methods and for each sample. Number of alleles calledand peak heights generally were consistent with DNA quantityyield as assessed by Quantifiler in most cases (Table 1). In 40 suc-cessfully typed bone samples extracted by the use of the Maxwell16 system, we obtained full profiles for 28 samples. Partial profiles

Table 1Nuclear DNA quantity and STR typing efficiency: Mean DNA concentrations and the number of successfully typed autosomal STRs for each bone sample for both extractionprocedures (QIAamp Mini columns and Maxwell 16 platform). Ct values noted as ‘‘>40’’ correspond to ‘‘undetermined’’ values.

Sample Age(years)

Sampletype

QIAamp Mini columns Maxwell

Mean DNA concentration(ng/uL)

Mean CtIPC

Mean Ctsample

STRloci

Mean DNA concentration(ng/uL)

Mean CtIPC

Mean Ctsample

STRloci

S1 15 Femur Below 0.023 27.95 32.42 5/16 0.121 26.02 32.48 16/16S2 65 Femur 0.0725 30.75 30.73 16/16 Below 0.023 26.04 35.49 14/16S3 65 Femur 0.972 27.55 28.87 16/16 0.861 26.14 29.71 16/16S4 17 Femur n.d. 26.35 >40 4/16 0.192 26.27 31.77 14/16S5 15 Femur n.d. 24.64 >40 1/16 0.0292 26.67 34.91 16/16S6 15 Femur 0.0307 28.29 31.96 16/16 Below 0.023 26.08 36.1 11/16S7 20 Femur Below 0.023 26.31 35.22 4/16 0.0435 26.06 33.82 16/16S8 15 Femur n.d. 26.04 >40 1/16 0.265 26.26 31.44 16/16S9 15 Femur Below 0.023 25.97 37.15 1/16 0.0689 26.25 33.18 16/16S10 15 Femur n.d. 26.18 >40 2/16 0.0863 26.21 32.86 16/16S11 15 Femur n.d. 26 >40 3/16 0.162 28.84 31.79 16/16S12 15 Femur n.d. 26.01 >40 1/16 0.835 27.81 29.63 16/16S13 15 Femur 0.0393 26.96 34.29 12/16 0.131 27.82 32.06 16/16S14 15 Femur n.d. 26 >40 1/16 0.124 26.05 32.39 16/16S15 15 Femur n.d. 25.84 >40 1/16 0.211 26.02 31.64 16/16S16 20 Femur 0.0553 26.24 32.3 16/16 Below 0.023 26.15 33.87 16/16S17 15 Femur 0.0637 26.15 32 16/16 Below 0.023 26.18 34.13 16/16S18 15 Femur 0.102 26.26 31.37 16/16 0.0671 26.32 31.95 16/16S19 15 Femur 0.164 26.37 30.4 16/16 0.063 26.45 32.03 16/16S20 20 Femur 0.0394 26.48 35.64 15/16 0.148 26.9 32.43 12/16S21 20 Femur 0.0504 26.59 33.5 16/16 0.228 26.77 31.8 16/16S22 20 Femur 0.383 26.28 29.6 16/16 0.107 26.16 31.37 16/16S23 20 Tibia 0.0457 26.62 32.55 16/16 Below 0.023 26.36 36.57 16/16S24 15 Femur n.d. 26.31 >40 4/16 Below 0.023 26.14 35.01 15/16S25 15 Cranial Below 0.023 26.85 37.01 7/16 n.d. 26.32 >40 6/16S26 15 Humerus Below 0.023 26.32 34.05 14/16 Below 0.023 26.25 33.65 14/16S27 15 Cranial n.d. 26 >40 5/16 0.0441 26.08 33.38 9/16S28 20 Femur 0.0622 26.16 32.12 16/16 0.0232 26.19 33.5 16/16S29 65 Femur 0.144 28.95 29.89 16/16 0.152 27.01 32.37 16/16S30 15 Femur 0.0827 26.39 31.67 16/16 Below 0.023 26.14 34.18 16/16S31 15 Femur 0.195 26.25 30.5 16/16 0.0518 26.31 32.31 16/16S32 20 Femur 0.121 26.33 31.16 16/16 0.0354 26.28 32.9 16/16S33 20 Femur 0.0327 26.43 33.02 16/16 Below 0.023 26.23 33.61 16/16S34 20 Radius Below 0.023 26.48 34.86 1/16 Below 0.023 26.3 37.38 1/16S35 20 Ulna Below 0.023 26.45 36.87 1/16 Below 0.023 26.41 38.06 1/16S36 20 Femur Below 0.023 26.35 35.47 5/16 Below 0.023 26.4 36.11 5/16S37 20 Femur Below 0.023 26.18 35.6 1/16 Below 0.023 26.2 36.37 1/16S38 19 Femur 0.0498 29.16 31.2 14/16 0.268 26.77 31.57 16/16S39 1 Femur 0.88 27 23.88 16/16 0.871 26.22 27.97 16/16S40 1 Femur Below 0.023 27.86 37.59 13/16 0.125 27.21 30.54 16/16

274 M. Karija Vlahovic, M. Kubat / Legal Medicine 14 (2012) 272–275

with up to four loci missing were obtained for five bones and forthe remaining seven bones less than twelve loci were amplified(Table 1). Full Identifiler Plus 16-STR genetic profiles were obtainedfrom 27 femurs and one tibia (Table 1). In five bones with partialprofiles (11 loci + Amelogenin), STR loci that were not amplifiedwere primarily the longest loci CSF1PO, D2S1338, D18S51 and FGA.

We did not observe artifacts like additional peaks either in thesample extracts or the negative extraction controls.

Fragments of radius, ulna and cranial bones gave the lowestsuccess of DNA typing regardless of the method used to extractDNA.

4. Conclusion

Nuclear STR analysis has been found highly successful to iden-tify skeletal remains from mass graves in Croatia [3,11,12]. Tofulfill this mission more than 2000 bone and teeth samples wereprocessed using manual DNA extraction procedures based onclassic organic extraction or silica based purification systems. Toreduce handling errors and increase a throughput [13–16] a perfor-mance of robotic platform was tested. The DNA extraction methodfrom old bones using Maxwell� 16 Forensic Instrument Systemgave quite satisfactory results. The DNA quantification results indi-cated that the robotic platform outperformed the manual columnextraction for twenty-one samples. When sufficient quantities of

DNA were extracted the reportable profiles were genotyped (85%of the samples). The Maxwell 16 system extraction procedureshowed a good inhibitor removal. Maxwell 16 platform can processup to 16 samples in less than 30 min and is a good option to use in-stead of the manual procedure.

Conflict of interest

The authors declare that they have no conflict of interest.

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