Fourth Annual DNA Grantees' Workshop
Monday, June 23, 2003
MORNING SESSION
GBI Experience with the NIST SRM 2392–I
Diana Williams
Biography
MR. FRANK: Our next speaker is Diana Williams. Diana is a forensic biologist with the division of forensic sciences at the Georgia Bureau of Investigation (GBI) in Decatur, Georgia. She received her bachelor of science degree in chemistry at the University of North Carolina and a master's degree in forensic science at the University of Alabama. Her research interests include molecular population substructure in forensically important flies using mitochondrial DNA analysis.
Today she's going to continue Barbara's [Levin] topic by discussing GBI's experience with the NIST (National Institute of Standards and Technology) standard SRM (standard reference material) 2392–I.
MS. WILLIAMS: Good afternoon. I'm just going to discuss a little bit about GBI's experience with SRM (standard reference material) 2392–I. NIST supplied us with all of the sequencing items. We received a tube of DNA containing HL–60 and all 58 sets of primers. We used the QIAquick PCR (polymerase chain reaction) purification kits, Big Dye Terminator cycle sequencing ready reaction kits, Edge Gel filtration cartridges for cleanup, and a 310 with Pop-6 polymer in the 47-centimeter capillaries. Williams: Slide 1 and 2
We used a 9600 thermocycler for amplification, followed the NIST protocol for amplification, and ran most amplification gels using 2.75 agarose, purified the PCR purification with the QIAquick kits, and did a postpurification recovery gel with the same 2.75 agarose. Williams: Slide 3
We performed the cycle sequencing with the Big Dye kit protocol and purified those with the Edge Gel filtration cartridges. Since I resequenced some of the samples with more amplicon, I did not get to purify them before precipitation because I ran short of the cartridges, but I was still able to get good results with those samples. Then I used isopropanol precipitation per the Big Dye kit instructions. Williams: Slide 4
We used the ABI (Applied Biosystems) 310 for sequencing and analyzing with the sequencing analysis software and then formed the comparisons using Sequencher. Williams: Slide 5
Doing this, we were unable to amplify the section using primer set 51. I attempted using more template but was still unsuccessful. As I understand it, there was a problem with that primer set, and, although it was modified, we did not get the opportunity to resequence it. Just for verification purposes I resequenced some of the samples. So I just listed how many times I had to do that. Williams: Slide 6
Currently at GBI, we're not performing any casework with mitochondrial DNA nor do we have any set protocols, so this was like flying by the seat of my pants when I was doing it. I would want to do some further validation with the techniques, especially with quantitation of the products, in order to get a better idea of how much I need to put in for cycle sequencing. Ultimately, then, I would not need to resequence as many samples as I did. Williams: Slide 7
Unfortunately, due to budgetary concerns, we're not going to do any mitochondrial DNA analysis in the immediate future. Given more time and some more materials, I would have liked to resequence some of the samples—considering our quantitation problems—to get cleaner data and higher peaks. Most of the time I underestimated the amount of the amplicon that I needed for cycle sequencing. Williams: Slide 8
Overall, I thought that the standard reference material was very easy to sequence, with no difficult areas, like Barbara said, with length heteroplasmy and C-stretches. For anybody who would like to see a good example of heteroplasmy, if they haven't encountered it before, the heteroplasmy at position 12071 was very clear and easy to see. Williams: Slide 9
MR. FRANK: Thank you very much.