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A new study in the Journal of Forensic Sciences assesses a kit made by a Japanese company that allows analysts to test small chips of bone, instead of powder. The kit allows the analysts to extract DNA samples without having to pulverize the bone. (Photo: Courtesy of Michelle Harrel)

When all that is left is bone, forensic scientists have an exacting, sometimes unforgiving, challenge. Depending on the condition of a skeleton, a dwindling set of information can be gleaned from what remains. DNA in particular can be difficult, since grinding interior parts of bones can lose material, and be a potential contamination source.

But a new study in the Journal of Forensic Sciences assesses a kit made by a Japanese company that allows analysts to test small chips of bone, instead of powder.

The Sam Houston State University team found that the TBone Ex kit, made by the Tokyo-based DNA Chip Research, Inc., effectively worked to extract DNA samples without having to pulverize the ossified material.

“This study demonstrates that digestion of whole bone fragments is an efficient alternative to powdering bones for DNA extraction without compromising downstream STR profile quality,” the authors write.

The team took six bone samples from human cadavers, cleaned and sanded them, and washed them with solution. Shavings of about 50 mg each were taken with a Dremel tool, and then demineralized and digested with the TBone Ex decalcification kit. Other control samples were traditional 50 mg powder samples isolated and then assessed through PCR purification columns made by Qiagen. 

Michelle Harrel, a Sam Houston State doctoral candidate and one of paper’s authors, corresponded with Forensic Magazine about the results this week, explaining the work. The following question-and-answer session is unedited.

Q: I wanted to know overall what the benefits are of using this TBone Ex kit, as opposed to powder. The paper mentions lower processing time, lower risk of contamination, and lower potential for material loss through the powdering process.

A: From my experience (other labs may use different methods/equipment and have different experiences), while transferring the powder from the grinding vial to a sample or long-term storage tube, some of the particles can become airborne and possibly contaminate other samples or be a breathing hazard for the processor. Additionally, some of the powder sticks to the inside of the grinding vial and can't be completely scraped out, leading to recovering less sample than was put in. Using a whole bone chip instead of powder virtually eliminates, or at least greatly reduces, these risks. In addition to those already mentioned, the bone sample is not totally destroyed with this system like it would be if it was powdered which results in sample preservation and the possibility for additional processing. Also, the buffers in this kit are pre-aliquoted in individual tubes - this removes additional preparation steps, making it simple and user-friendly.

Q: As far as the drawbacks, the paper mentions that automated platforms lead to lower DNA concentration and lower STR profile quality. Does this mean that combining the TBone Ex with a manual process produces the best results?

A: Compared to performing the DNA extraction with an automated system, the manual process consistently resulted in higher DNA yields. Additionally, we observed that using the TBone kit with a manual process gave similar results compared to the same manual method alone with powdering, or to put it another way – using the TBone kit with non-powdered bone did not negatively affect DNA yield. Another drawback though might be that it has longer incubation times compared those already established with some other DNA extraction kits for powdered bone.

Q: These were relatively new samples, less than six years old. Would more material be required for older bones?

A: Potentially, yes. However, I believe it really depends on the condition of the bone(s), how well it was preserved, and what type of downstream analysis will be used (ex: nuclear vs mito. typing), among other factors.

Q: What kinds of bones were used? Does that matter? (I know a lot of the fossils from ancient anthropological sources I write about are thousands of years old, and they have come to rely on the inner ear bones, as opposed to the inner parts of the biggest and thickest bones).

A: We focused on long bones (humerus, femur, tibia) since those are most commonly processed in laboratories due to the amount of starting material required (up to 2 g) for many protocols. The only physical limitation with this kit is that the bone sample has to fit in a 50 mL conical tube and be completely submerged in the buffer. Since we used a very small amount of bone (50 mg), we did not have any issues regarding that.

Q: Does this mean you can get more DNA from non-traditional bones using the TBone Ex kit?

A: Although we have not looked into it and I haven't seen/read other reports using the TBone kit for non-traditional bones, many studies using other methods have shown that other non-traditional bone sources may have similar or higher amounts of DNA. So basically, I'm not sure but I assume it would at least make it easier/simpler to do so.

Q: What is the best application of this technology, and methodology?

A: One of the best applications, in my opinion, is when there is a limited amount of bone sample available for testing. The buffer system in the TBone kit allows for an intact piece of bone to be digested prior to DNA extraction and still be available for additional testing if needed, rather than being consumed in a single extraction. Another beneficial application would be the processing of historical/museum remains as the bone(s) can potentially be digested, rinsed, and returned with little or no visible damage.

Q: I think that’s about it, to start with? Just one question about you: are you focused primarily on bone work, or do you have publications on other analyses?

A: My PhD dissertation (which I am currently working on) is all centered around improving methods that are involved in the identification of skeletal remains. I have given presentations over other analyses, but as of right now, this is my only journal publication.

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