Application of Non-powdering Sample Pre-Treatment Methodfor Bone and ToothTakanashi K, Takagi Y, Ueda Y, Hata J, Ishizawa Y, Lim CRDNA Chip Research Inc., 1-15-1 Kaigan, Suzue Baydium 5F, Minato-ku, Tokyo 105-0022, Japan

Introduction

ResultIdentification of human remains using DNA extracted from tooth or bone is common in forensic science. Extraction of DNA from hardtissues requires pulverization in an extremely well controlled environment to prevent cross-contamination of powdered samples. To
overcome this issue, we introduce a liquid-based sample pre-treatment reagent, Tbone EX Kit. Extraction of DNA directly from a whole
tooth or a piece of bone just requires sequential treatment with three types of solutions in the kit, bypassing the problematic pulverization
step. In this study, we have performed STR genotyping of a tooth sample, extracted using Tbone EX Kit coupled with different purification
TMmethods including PrepFiler BTA. We have also attempted to extract DNA of ancient cremated bone sample and would share theexperience and knowhow.

Experiments and Results
1. Experiment Flow

2. ToothPhenol Treatment
105One tooth and two ancient cremated bone samples
were treated with Tbone EX Kit without pulverization
respectively.

175245315385455

105

6000

6000

4000

4000

2000

2000

00Fig. 1
Male individual, 40sSample weight 2.1g
Wisdom tooth, decayed, nerve removed
Kept at room temperature for about one year

Table 1245

315385455

DNA yield and STR Loci of tooth sample
Phenol Treatment

1st Round
2nd Round

Decalcification

175

STR genotyping graphs of tooth sample 2nd round extraction.

Tooth sample

Tbone EX Kit

No Phenol Treatment

No Phenol Treatment

DNA content Loci(ng/ul)

DNA content Loci(ng/ul)

0.000186
0.039

0.000875
0.0389241624

STR Amplification was performed with GlobalFilerTM PCR Amplification Kit, data
was analyzed and visualized using GeneMapperTM ID-X v1.4. All 24 STR lociwere detected from DNA extracted at second round.

Protein Digest
Appearance

Contents

3. Ancient Cremated BonesThe whole tooth/bone was dipped insolution A and processed according tothe manual (Tbone)

Sample 1:cremated male individual ( ~ 1915 ), 50s, sample weight 1.38g
Sample 2:cremated male individual ( ~ 1867 ), 2 years old, sample weight 1.05g
105175245

315

385

455

105

160

160

00175

245315385

455

Phenol/Chloroform
Treatment or Not

PrepFiler

TMBTA

Sample 1

Sample 2

Fig. 2

STR genotyping graphs of ancient cremated bone samples.

Table 2DNA yield and STR Loci of ancient cremated bone samples
DNA content Loci(ng/ul)

DNA purification
Sample 1
Sample 20.000494
0.000198

46Conclusions
1. As pulverization of samples is not required, Tbone EX Kit reduced hands-on time required for extraction ofDNA from bone and tooth.
2. Qualities of DNA extracted is good enough for down stream forensic analysis such as STR genotyping.
3. Extraction of DNA from cremated ancient sample is a difficult task especially when region containing DNA has
been burnt. However, we could still able to extract from regions where cremation is partial or not complete.

Acknowledgements
Authors would like to thank Dr. Shinji Harihara from University of Tokyo, Dr. Hitoshi Sumi from University of Tsukuba for ancient bone samples andThermo Fisher Scientific Inc. for reagents and STR genotyping operation.