Take a sneak peek at the new NIST.gov and let us know what you think!
(Please note: some content may not be complete on the beta site.).

View the beta site
NIST logo
Bookmark and Share

26plex Autosomal Short Tandem Repeat Assay


A short tandem repeat (STR) multiplex assay was developed with 25 autosomal loci plus the sex-typing locus amelogenin for a total of 26 amplified products in a single reaction. Primers for the loci were designed so that all of the amplicons present are distributed from 65 base pairs (bp) to less than 400 bp within a 5-dye chemistry design with the 5th dye reserved for the sizing standard. A multiplex design strategy was developed to overcome challenges encountered in creating this assay. The limits of the multiplex were tested, resulting in the amplification of a wide range of genomic DNA sample concentrations from 2 ng to 100 pg with 30 cycles of PCR. The 26plex has the potential to benefit the forensic community for reference sample testing and complex relationship evaluation.


Multiplex polymerase chain reaction (PCR) is commonly used for forensic DNA typing. Commercial short tandem repeat (STR) assays amplify the Combined DNA Index System (CODIS) STR loci that are required for data entry into the national level of the U.S. DNA database. Additional loci can play a useful role in the forensic community in situations involving relatives such as missing persons or mass disaster victim identification, immigration testing with limited reference samples, missing parentage testing which is often needed if only one parent and child are tested, and in cases involving incest.

A single amplification 5-dye multiplex PCR assay has been developed to combine 25 autosomal STR loci plus the sex-typing marker amelogenin in one reaction to enable rapid analysis of reference samples. These new STR loci were described previously as mini-short tandem repeat (miniSTR) loci (Hill et al, Coble et al) and were selected because they span unused chromosomal locations across the 22 autosomes. They have been characterized so that they may be combined without conflicting with the current 13 CODIS loci.

These new STR loci have been examined in U.S. population samples and sequenced for calibration of allele nomenclature in standard samples. In addition, a concordance evaluation was performed with the multiplex to compare genotypes obtained with the previously characterized miniSTR loci to determine if there are any null alleles present with the newly designed primer sets. The multiplex was run across father/son sample pairs to determine the individual mutation rates of each STR locus characterized. An extended family sample study was performed to determine the effect on likelihood ratios when adding additional loci to the analysis. The results from these studies were reported in Butler et al.

Challenges in designing and building a large multiplex required multiple iterations of the design due to the incompatibility of some primer sets. PCR primers were considered failures and candidates for loci redesign if the following occurred: incomplete adenylation, presence of artifacts, low signal, non-specific products, or absence of PCR product. Other issues encountered and requiring multiple trials included the elimination of troublesome loci and redesign of incompatible primers, empirical balancing of primer concentrations for locus-to-locus balance, and fluorescent dye-artifact removal with post-amplification filters. Fundamental steps involved in building this multiplex included the initial multiplex design, the strategy for primer design, primer-to-primer inter-comparisons, and necessary primer modifications made to the original designs.

Certified and reference values have been assigned to all alleles of the 12 components of SRM 2391b for the 26 additional loci, and the Certificate of Analysis updated. Use of this SRM enables calibration of all genotypes resulting from analysis with the 26plex.

Major Accomplishments:

  • Design of a 26plex autosomal STR assay to aid human identity testing
  • Certification of SRM 2391b for the 26 STR loci

Start Date:

March 1, 2007

End Date:

October 2008

Lead Organizational Unit:


Source of Extramural Funding:

National Institute of Justice (NIJ) funded the work described here through an interagency agreement with the NIST Office of Law Enforcement Standards.