Pyrosequencing Primer Design for Forensic Biology Applications

The polymerase chain reaction (PCR) is used to copy DNA in vitro for a variety of applications including amplifying a target DNA, mutating a base, adding tags, and sequencing by synthesis applications. Next-generation sequencing (NGS) is a DNA sequencing technology that has been applied to screening cancer and tissue variants, deep sequencing, and gene expression analysis, and more recently, it has been applied to DNA typing for human identification, estimating age, and detecting and differentiating body fluids. Body fluids are normally identified using color tests, microscopy, and immunochromatographic assays. Pyrosequencing is an NGS approach that has been applied to body fluid analysis. The pyrosequencing assays can detect one or several mixed body fluids by analysis of their tissue-specific differentially methylated regions (tDMRs). Here, the process of designing pyrosequencing primers for forensic biology applications is described.