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Development of Miniplex Primer Sets for the Analysis of Degraded DNA, Final Report

NCJ Number
Date Published
85 pages
This is the final report on a project to develop a new set of multiplexed PCR reactions for the analysis of degraded DNA.
Currently, forensic laboratories confronted with highly degraded DNA move toward mtDNA sequencing, which has a higher likelihood of producing a result than genotyping with nuclear DNA. Unfortunately, because mtDNA are haploid markers, the admixing that occurs with sexual reproduction does not occur, so the statistics for determining identification are not nearly as definitive as with STRs and nuclear DNA. DNA markers known as miniplexes use primers that have shorter amplicons for use in STR analysis of degraded DNA. The project described in this report defined six new STR multiplexes, each of which consists of three to four reduced size STR loci, each labeled with a different fluorescent dye. Reductions in size of up to 300 basepairs are possible with these new amplicons. To demonstrate compatibility with commercial STR kits, a concordance study of 532 DNA samples of Caucasian, African-American, and Hispanic origin was conducted. A 99.77-percent concordance between allele calls with the two methods was achieved. In the 532 samples, there were 15 samples that showed discrepancies at 1 of 12 loci. Such uncommon deletions can be expected in certain samples and will not affect the use of the miniplexes as tools for degraded DNA analysis. This report describes a number of studies conducted to determine how the miniplexes performed under various conditions. The results show that miniplexes performed well at template concentrations above 125 pg and displayed excellent sensitivity for low copy number DNA. Overall, the results showed a greatly improved efficiency in the analysis of degraded DNA when compared to commercial STR genotyping kits and confirmed that smaller PCR amplicons provide an attractive alternative to mtDNA for the forensic analysis of degraded DNA. Appended protocols and 34 references

Date Published: January 1, 2005