No More Powdering:
DNA Extraction Pre-treatment Kit for Teeth and Bones
Nor Alfarizan Mokhtaruddin1, Mohd Iqbal Zulkafli1, Mohammad Azhar Mat Zin1, Nor Aidora Saedon1, YukoTakagi2, Lim Chun Ren2, and Cornelia Charito Siricord1
1Department of Chemistry Malaysia, Selangor, Malaysia ; 2DNA Chip Research Inc., Tokyo, Japan

Introduction

Results

Successful DNA extraction from teeth and bones is crucial for identification ofhuman remains. DNA extraction from hard tissues inevitably requires crushingand grinding of samples into powder, but such procedure is troublesome andprone to cross-contamination. Tbone EX Kit is a DNA extraction pre-treatment
reagents, which enable extraction of DNA directly from whole tooth or bone,
bypassing the step of powdering (Fig.1C). Extracted DNA can be used fordownstream assays such as mitochondrial DNA sequencing and STR
genotyping.

Concentration of purified DNA obtained by two methods are shown in Table 1.
Tbone EX Kit was compatible with Applied Biosystems PrepFiler™ BTA andAutoMate Express™ DNA Extraction System, facilitating successful DNA
extraction from bones without the need of powdering. Tbone EX Kit yieldedhigher concentration of DNA than conventional protocol in all thirteen samples
tested. Samples A201, A200 and A259 did not yield detectable DNA by freezer
mill protocol, but all three resulted in measurable amount of DNA by pretreatment with Tbone EX Kit combined with PrepFiler™ BTA and AutoMate
Express™ DNA Extraction System.

ABCFig. 1 Appearanceof Tbone EX Kit. (A)Kit container. (B)Contents of the kit.
(C) Example ofDNA extraction froma whole tooth.

Tbone EX Kit is widely used by Japanese forensic laboratories in police force
and universities. However, its compatibility with downstream DNA extraction
systems other than QIAamp® columns from QIAGEN has not been examined
extensively. At Department of Chemistry Malaysia, we have evaluatedapplicability of Tbone EX Kit combined with downstream Applied Biosystems
PrepFiler™ BTA and AutoMate Express™ DNA Extraction System.

Experimental Procedure(A) Freezer mill(B) Tbone EX Kit

1. Sample cleansing

Table 1. Amount of starting materials and concentration of purified DNA from two extraction
methods. (A) Powdering with freezer mill. (B) Direct incubation of bone piece with Tbone EX Kit.

Treatment(A) Freezer mill(B) Tbone EX Kit

SampleMarkingStarting
amountInput after
powdering

Quant result(ng/ul)Starting
amountQuant result(ng/ul)

A201

0.5 g

70 mg

0.00000.5 g

0.0011

A186C

0.5 g

70 mg

0.00020.5 g

0.0004

A200

0.5 g

70 mg

0.00000.5 g

0.0014

A192

0.5 g

70 mg

0.00050.5 g0.0018

A198

0.5 g

70 mg

0.00160.5 g

0.0143

A184a

0.5 g

70 mg

0.00070.5 g

0.0097

A265

0.5 g

70 mg

0.00180.5 g

0.0121

A261

0.5 g

70 mg

0.00350.5 g

0.0227

A267

0.5 g

70 mg

0.00190.5 g

0.0102

A259

0.5 g

70 mg

0.00000.5 g

0.0032

A273

0.5 g

70 mg

0.0031

0.5 g

0.0049

A274

0.5 g

70 mg

0.02980.5 g

0.0367

A271

0.5 g

70 mg

0.14100.5 g

0.3150Conclusion
Freezer mill

2. Powdering

2. Decalcification
Tbone
EX KitPrepFiler BTA
Extraction Kit
on AutoMate
Express™
DNA Extraction
System

3. Protein digest

3. Protein digest

4. DNA extraction

4. DNA extraction

5. DNA purification

5. DNA purification

PrepFiler BTA
Extraction Kit
on AutoMate
Express™
DNA ExtractionSystemFig. 2. Experimental flow of two sample treatment methods. (A) Powdering with freezer mill.
(B) Direct incubation of bone piece with Tbone EX Kit.

Thirteen bone samples were cleaned and cut into convenient size prior to theexperiment. For protocol (A), the samples were crushed into powder byfreezer mill, and DNA was extracted according to the standard procedure ofApplied Biosystems PrepFiler™ BTA. For protocol (B), intact pieces of bones(0.5 g each) were incubated with reagents of Tbone EX Kit to carry out
decalcification. Samples were treated by following the standard procedure ofTbone EX Kit up to the protein digest step, then by PrepFiler™ BTA
procedure thereafter.

Tbone EX Kit was developed for extraction of DNA from hard tissues,
bypassing troublesome step of powdering. Here we demonstrated that the kit
indeed enables extraction of DNA from non-powdered bone samples. We also
evaluated compatibility of the reagent with Applied Biosystems DNA extraction
system, and observed that it is not only compatible, but also increases theyield of DNA, comparing to the conventional method. Tbone EX Kit can bebeneficial in reducing the handling time to minimize the risk of crosscontamination, while increasing the chance of obtaining DNA from previously
challenging samples. Our data showed that Tbone EX Kit can be incorporated
into existing forensic DNA automated extraction system. Tbone EX Kit also
allows extraction of large number of samples in parallel by simply pretreating
the samples by dipping them in the solution. This potentially saves time andeffort, while increasing the overall efficiency and quality of DNA purificationfrom hard tissues.

Acknowledgement
Authors would like to thank Mr. Chin Kah Lock from Analisa Resources (M) Sdn Bhd for
coordinating the collaboration between Department of Chemistry Malaysia and DNA Chip
Research Inc.

Reference:
Evaluation of a new experimental kit for the extraction of DNA from bones and teeth using a nonpowder method. Leg Med (Tokyo). 2010 Mar;12(2):84-9. Epub 2010 Jan 27.

Tbone EX Kit is manufactured by DNA Chip Research Inc. under patent license agreement with Shinshu University and Hitachi Solutions, Ltd.