DNA extraction and amplification from formalin fixed paraffin embedded tissues

ABSTRACT

Tissue preservation is very important in clinical settings for subsequent histological studies or genetic diagnosis. It is also critical in forensic investigations where human remains are collected for positive identification following plane crashes, terrorist bombings, homicides, and mass murders. Historically, formalin fixed [FF] tissues could not be used as a source of DNA in forensic science due to the fact that the DNA was too degraded for DNA analysis. With the introduction of the polymerase chain reaction [PCR] technique to forensic science, the usefulness of DNA from this biological material has been re-evaluated. This study attempts to evaluate the applicability of formalin fixed paraffin embedded [FFPE] tissue fit into the field of forensic science by examining these tissues as a source of DNA and applying current methodology. The study was conducted on 35 formalin fixed paraffin embedded [FFPE] different tissue samples [liver, kidney, lymphoid tissue, colon] prepared by and received from the Pathology Department at the Alexandria Faculty of Medicine. These tissues were removed at autopsy and put in 10% buffered neutral formalin [BNF] for a period of 24 to 48 hours. The tissues were then processed in an automated tissue processor. The samples were stored at room temperature for 24 months before processing. After getting rid of paraffin by using ParaClear reagent, the DNA was extracted by the organic phenol-chloroform method. The quantity and quality of the extracted DNA was evaluated by running a yield gel. The exact concentration of the extracted DNA was determined using the QuantiBlot[R] Human DNA Quantitation Kit. The extracted DNA was amplified for 3 forensic PCR systems the AmpliType PM+DQA1, AmpliFLP D1S80, and The AmpFlSTR Profiler Plus, the three systems include 16 PCR loci; HLA DQA1, LDLR, GYPA, HBGG, D7S8, GC, D1S80, D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820 and amelogenin. The first six, HLA DQA1, LDLR, GYPA, HBGG, D7S8, and GC are typed by reverse dot blot, D1S80 is an amplified fragment length polymorphism [AmpFLP] system and the others are short tandem repeats [STRs]. This study showed that the DNA extracted from formalin-fixed, paraffin-embedded tissues is usually of adequate quality for PCR amplification using well characterized forensic PCR systems