Novel STR Multiplexes with Reduced Size to Analyze Degraded DNA

The goal of this project is to develop novel STR multiplexes with reduced amplicon sizes for the analysis and detection of degraded and contaminated DNA. In such samples, DNA template can become highly fragmented due to bacterial, biochemical, or oxidative processes, and the possibility of finding an intact target sequence for PCR is greatly reduced. In this project researchers are developing and validating the use of STR Miniplexes. These are CODIS STRs in which the primer sequences are moved as close as possible to the repeat region. A set of four miniplex kits have been developed covering 12 of the 13 STRs. In a series of studies with enzymatically degraded DNA researchers have demonstrated that reduced size STRs improve the success rate when degraded template is used. Allele dropout is minimized and amplification efficiency improves. Investigators have also performed a concordance study of over 540 samples. Of these samples, 16 discrepant samples were found, all but one of which occurred at vWA or D13. Several of these samples were sequenced, and the results show interesting effects due deletions or substitutions at the primer binding site. Researchers have also applied these primers in the analysis of human skeletal remain samples (n = 31) that have been exposed to a variety of environmental conditions. These samples were obtained from the Forensic Anthropology Center (FAC) at the University of Tennessee in Knoxville and the Franklin County Coroner’s Office in Columbus, Ohio. These samples provided a method to examine the efficiency of PCR amplification by the Miniplex primer sets using inhibited and degraded DNA templates. The amplification efficiency of these primer sets was then compared to the PowerPlex® 16 system. Since many of the samples which will be analyzed using miniSTRs will also contain PCR inhibitors, the study has examined the effect of these substances on PCR amplification with Miniplex sets. The effects of these substances on the PCR reaction can vary from different levels of attenuation to complete inhibition. Researchers studied the effect of varying concentrations of PCR inhibitors using both Miniplexes and larger STR amplicons. In general the project found that while amplification efficiency improves with shorter STRs, the effect of PCR inhibition was the same. As a result of this work, various template cleanup steps were investigated, including spin columns, BSA and low melting agarose. [Notes:] Butler, J.; Shen, Y.; McCord, B. The Development of reduced size STR amplicons as tools for analysis of degraded DNA. Journal of Forensic Sciences, 2003, 48(5), 1054-1064. Chung, D.; Drabek, J.; Opel, K.; Butler, J.; McCord, B., A study on the effects of degradation and template concentration on the amplification efficiency of the STR miniplex primer sets, J. Forensic Sciences, in press. Drábek, J.; Chung, D.; Butler, J.; McCord, B. Concordance study between miniSTR assays and a commercial STR typing kit, J. Forensic Sciences, in press.