Nucleotide sequence analysis to identify a one-step mutation in a STR DNA profile during paternity testing at locus D7S820

Abstract

During routine paternity testing with AmpFℓSTR^® Identifiler^® Plus Kit™, the kit failed to amplify the child’s allele at locus D7S820 leading to parent-child inconsistency with a single-step mutation. The aim was to identify possible causes of this mismatch. New singleplex primers were designed and the samples were amplified, cloned and sequenced using pJET1.2/blunt cloning vector forward and reverse sequencing primers. The amplicons were ascertained using CLC Bio Main Workbench. We confirmed the presence of allele dropout at the child’s locus. We describe a single nucleotide polymorphism (SNP), from cytosine (C) to thymine (T) in the kit primer binding site region of the alleged father’s profile. The child’s profile changed from homozygous to heterozygous showing that the commercial kit failed to amplify the allele and this was concluded to be most likely due to polymerase slippage.