Reparation of degraded DNA improves PCR amplification of larger STR loci

Abstract

Postmortem tissues frequently produce deficient STR profiles, which is a problem in forensic identification and paternity testing. This is because traditional STR typing samples can be affected by inhibitors that slow or inactivate the PCR reaction, and by DNA degradation that often leads to allele dropout and poor PCR amplification of the larger sized STR loci. The formaldehyde commonly used to fix soft tissues generates DNA degradation, due to single and double strand breaks. Likewise skeletal human remains are usually inadequate for PCR amplification since the DNA is often degraded due to exposure to heat, humidity, light and microorganisms. Therefore in order to improve the genotyping of traditional STR from degraded DNA samples using a simple approach, the objective of this work was to partially restore the DNA length by filling the single strand breaks before the PCR amplification. Formalin fixed soft tissues and bone remains were subjected to traditional DNA purification with solvent extraction and Amicon ultra filtration. The purified DNA was submitted to a restoration treatment involving a pre-polymerase chain reaction assay involving a incubation with dNTPs and Taq DNA polymerase at 72 °C for 20 min, followed by a denaturing step before the PCR amplification using the AmpFlSTR Identifiler kit. The pre-PCR reparation treatment of the DNA allowed to amplify the larger STRs loci that were missing in the untreated samples. Therefore, in situations where standard STR kit fails, the pre-PCR DNA repair increases the probability of obtaining a full DNA profile.