Rachel Kieser, former PhD student at the University of North Texas Health Science Center, shares the poster that she presented at ISHI on a new method to target degraded DNA. This method was used to applied to skeletal remains over 850 year old from the royal House of Aragon, showing it’s applications for more than just casework.
Reverse Complement PCR (RC-PCR) is a novel one-step PCR target enrichment technology for amplifying short fragments of DNA. It allows for simultaneous amplification and tagging of a targeted sequence construct in a single, closed tube assay. The library-prepared products are suitable for massively parallel sequencing (MPS).
Travis: Good morning Rachel. It’s good to see you! How are ya?
Rachel: I’m good. How are you?
Travis: I’m doing great, thanks. Hey, I was wondering, do you think you could share here what you’ve brought to ISHI 30?
Rachel: Absolutely. Well this is just one of them, but this is the method and the other one’s more of an application. So, this is a brand new, novel, PCR-targeting enrichment system that this company in the Netherlands had produced, but for a clinical application. I kind of just emailed them on a whim and was like, “This is what I want to do for forensics. Let’s see what we can do.” We came together to make this assay that is really good at targeting really fragmented DNA, so it’ll be really helpful in your degraded and damaged DNA, essentially. So, we designed an assay that targets 27 identity SNPs, so it’s a start. It’s not all the way there yet, but it’s a start. It’s performing phenomenal. So, it works on kind of this reverse compliment system. So, inside your tube, you basically make your targeted primers. So, one of the big issues or disadvantages of normal library prep is that there are a lot of PCR amplification steps. There’s a lot of room for contamination, which you don’t want with your degraded DNA. Here, it is a one step-you just put your DNA in a tube, put it on the thermocycler and prepare it for library preparation. So, everything is simultaneously tagged, targeted, and amplified in one closed-tube system, which is incredible for forensic scientists. So, I really wanted to bring it to ISHI to just highlight how amazing it is. So, here is a method. It probably goes into a lot more detail than I probably can now, but anyone stop by, and I’ll explain it.
And here, we just kind of did a validation or an evaluation of the system to see how it works with sensitivity. We went down to 60 picograms of DNA, which is about 10 cells of nuclear DNA, so it performs really well. And then we tried some known inhibitors, because everyone has problems with inhibitors and PCR, and it’s actually performing beautifully for most of them, especially calcium and collagen, which are in bone. It’s so phenomenal for highly degraded DNA.
And then, we tried some casework samples from our Center for Human ID, and it really helped gain some additive genetic information in addition to the STRs, so it does have a very advantageous effect. So, if anyone would like more information, also we applied it to the royal House of Aragon, skeletal remains, so check out that poster as well. There’s some mito thrown in, but there is nuclear, and I hope to see everyone.
Travis: How old were the bones that you used to do this testing?
Rachel: The royal house dates back to about 850 years to 950 years ago, so really old remains.
Travis: And you got some hits off of that?
Rachel: We did, we got some data! So, that is a huge advancement for us and it’s really exciting.
Travis: Thank you so much for sharing all of that with us. Thank you!
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