Jan 25 2016
Two Novel Multiplex INDEL Assays for use with Degraded DNA Samples
Most commercially available STR kits yield amplified fragments with lengths between 100 and 500 base pairs. However degraded biological samples typically are fragmented into pieces of approximately 180-200bps. When samples have been degraded to fragments less than 500 base pairs, many of the loci in STR amplification kits, fail to yield amplification products. INDELs are insertions or deletions of a DNA sequence, as determined by comparison to a known consensus sequence and have been suggested as a viable option for typing degraded samples. Their amplicons can be designed to be very small (~55bp as limited by PCR). As a non-repeating polymorphism, slippage artifacts are non-existent. INDELs have amplification products that differ in length and therefore can be run/ analyzed by capillary electrophoresis. This is another important feature when relating to their use in forensics since the laboratory infrastructure required to genotype INDELs is already present and does not require any next generation technology or development of special technology.
Written by: Kelly Sage and Sarah Sturm, University of North Texas Health Science Center
We became quick friends with the graduate student before us, Lindsey Thompson, who told us about the work she was doing with developing the AIM marker panel. She asked us for a couple extra sets of hands to help her with her project, and we became more interested in the INDEL system. Working in the forensics field, we saw the utility of having a marker system that would work well with amplifying degraded and low template DNA samples. We approached Dr. LaRue, Lindsey’s major professor, and asked if he had any related projects for us. He had the HID panel and the AIM panel for both of us to develop a multiplex.
Based on previously chosen HID and AIM INDEL marker panels, we designed unlabeled primers to amplify those specified regions. We then ran each primer pair in single on the Agilent 2200 TapeStation to check that the primers would in fact work and that no problems were seen. After that, the primer pairs were placed in groups of 5, trying to space out the base pair product size to 10 bp apart, for an initial multiplexing reaction that was also run on the Agilent TapeStation. Fluorescent-labeled primers are being ordered to run on the CE to provide more accurate and clear results. Running the primers in single showed that all 49 HID INDEL primers, and 29 out of 30 AIM INDEL primers that were designed were working. The initial multiplexes on the Agilent TapeStation showed that the primers are able to be multiplexed together and produce more informative results.
We are introducing two multiplex kits including one that could prove useful in missing persons cases. Click To Tweet
Since there are no kits available for these specific INDEL systems, we are introducing two multiplex kits into the field. Both are great choices to use for degraded or low template samples. One kit being for human identification, which could prove useful for not only comparing a suspect’s reference sample to an evidentiary sample but also in missing person cases which is the silent mass disaster around the world. The other panel is for ancestry informative markers, which can provide investigative leads for detectives involved in the case.
Our next step is to use the fluorescent labeled primers on the Applied Biosystems 3500 Genetic Analyzer and perfect the multiplex system to ensure the best results possible are seen. We will then perform population studies using the designed multiplexes.
The key collaborators on this project are Kelly Sage, Sarah Sturm, Frank Wendt, Lindsey Thompson, Jonathan King, Dr. Bruce Budowle, Dr. Bobby LaRue.
Kelly Sage grew up in Big Spring, Texas. She is currently earning her Masters of Science degree from the University of North Texas Health Science Center. She has a Bachelor’s Degree in Genetics from Texas A&M University.
Sarah Sturm is currently earning her Masters of Science degree from the University of North Texas Health Science Center. She has a Bachelor’s Degree in Chemistry from Fairleigh Dickinson University in New Jersey, where she is from.
WOULD YOU LIKE TO SEE MORE ARTICLES LIKE THIS? SUBSCRIBE TO THE ISHI BLOG BELOW!