Validation of a single tube fluorescent multiplex assay for simultaneous typing of 20 Y-STR loci

This paper describes a single tube assay for 20 fluorescent labelled Y-STR loci, which is rapid and robust state-of-art multiplex system. Reaction conditions, including annealing temperature, concentration of primers, magnesium and template DNA, DNA polymerase and reaction volume, were optimised to yield robust amplification. The assay could withstand moderate fluctuations in reaction parameters with no affect on haplotype analysis. Sensitivity and robustness of the multiplex system is revealed from the examination of different matrices and environmental conditions wherein complete haplotypes could be obtained in all the samples with no failures except those exposed to severe environmental stresses. Genotyping of the 20 Y-STR loci showed that all loci included in the multiplex are highly polymorphic in the Indian populations and its inclusion increases the discriminatory power of the marker system. It could be summarized that the developed Y-STR multiplex assay is a simple, sensitive and robust system highly suitable for concurrent analysis of 20 polymorphic Y-STR loci.