A Real-Time PCR Protocol to Determine the Number of Amelogenin (X–Y) Gene Copies From Forensic DNA Samples

Abstract

We present a fluorogenic real-time polymerase chain reaction (PCR) procedure to target a segment (106-112 base pairs [bp]) of the X-Y homologous amelogenin gene by measuring the 5' nuclease activity of the Taq deoxyribonucleic acid (DNA) polymerase using two (X-Fam-labeled and Y-Vic-labeled) specific Taqman MGB probes to enable simultaneous the detection of two specific PCR products (AMGY: 112-bp and AMGX: 106 bp) as they accumulate cycle by cycle during PCR. The method makes possible not only human nuclear DNA quantitation but also sex determination and has been applied to the analysis of low copy number DNA samples in forensic and ancient DNA studies. Specific quantification of human nuclear DNA is a recommended procedure in forensic casework as a way to adjust the DNA input on subsequent end-point PCR-based DNA typing approaches ensuring the optimal use of the limited amounts of nuclear DNA found in many forensic evidences. Nuclear DNA quantification also aids in the interpretation of the consistency of multiplex STR profiling data obtained from low copy number DNA samples.