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A New 26plex Autosomal STR Assay to Aid Human Identity Testing*(dagger)



Carolyn R. Steffen, John M. Butler, Peter Vallone


Multiplex polymerase chain reaction (PCR) has become the standard in forensic testing. Currently there are two commercial multiplex PCR amplification kits available that simultaneously amplify 16 short tandem repeat (STR) loci that include the 13 FBI defined CODIS markers. A new multiplex assay has been successfully designed and developed with 25 non-CODIS markers plus the sex-typing marker amelogenin for a total of 26 loci in a single PCR reaction. This novel multiplex represents the most autosomal STR forensic markers combined together effectively in one assay. All of the markers present are distributed from 65 base pairs (bp) to less than 400 bp within a five-dye chemistry design with the fifth dye reserved for the sizing standard. A genomic DNA sample concentration range from 2 ng to as low as 100 pg can be amplified efficiently with 30 cycles of PCR. The strategy for constructing this multiplex, including the initial selection and placement of loci, primer design and optimization, final empirical balancing and testing, as well as the many challenges encountered throughout the development process are reviewed. The present 26plex has the potential to significantly benefit the forensic community for reference sample testing and complex relationship evaluation including kinship analysis, criminal paternity testing, parentage testing, immigration testing, and for use in missing persons/mass disaster cases.
Journal of Forensic Sciences


forensic science, short tandem repeat, DNA, miniSTR, multiplex, non-core loci, NC markers


Steffen, C. , Butler, J. and Vallone, P. (2009), A New 26plex Autosomal STR Assay to Aid Human Identity Testing*(dagger), Journal of Forensic Sciences, [online], (Accessed May 22, 2024)


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Created August 31, 2009, Updated February 19, 2017