Jornal de pesquisa forense

Forensic DNA Typing of Old Skeletal Remains Using AmpFlSTR® Identifiler®PCR Amplification Kit

Abstract

Mian Sahib Zar, Ahmad Ali Shahid, Muhammad Saqib Shahzad, Kyoung-Jin Shin, Hwan Young Lee, Muhammad Israr, Eun Hye Kim, Zia Ur Rahman and Tayyab Husnain

Background: In this study DNA typing of old skeletal remains was improved through AmpFlSTR®Identifiler®PCR amplification kit using different approaches. Methodology: DNA extraction was carried out by silica columns based total demineralization extraction method.DNA quantification was carried out by Real Time PCR. DNA amplification was carried out by using AmpFlSTR®Identifiler® PCR amplification kit with modified conditions. Capillary electrophoresis and Data analysis were carried out by Genetic Analyzer 3130 (ABI) and GeneMapper ID software version 3.2. Results: DNA was detected in 17 out of 24 skeletal remains. Among them, in 7 samples DNA was in the range of 1-10 pg/µL, in 4 samples it was in the range of 22-69 pg/µL and in 6 samples the DNA was in the range of >100 pg/ µL. The CT value (<30) of 40 cycles indicated that the PCR inhibitors were removed during DNA extraction method. Promising results were obtained by increasing the number of PCR cycles from standard 28 to 33 instead of 32 in PCR reaction. Finally it was observed that consensus approach produced reliable and reproducible DNA profiles from old skeletal remains. Conclusions: Forensic DNA typing of old skeletal remains, through a multiplex AmpFlSTR®Identifiler® PCR amplification kit, is improved by using: a highly effective DNA extraction method, modified and optimized PCR conditions, increasing sensitivity of PCR amplification and consensus approach