Extraction and Analysis of Human Nuclear and Mitochondrial DNA from Electron Beam Irradiated Envelopes

Abstract

The United States Postal Service is considering methods such as electron beam irradiation to neutralize biological agents sent through the mail. While this is proven to reduce/eliminate pathogenic organisms, it may also degrade human genomic DNA and therefore hinder the ability to garner forensically informative genetic profiles. To determine the effects of electron beam irradiation on DNA typing, 16 white, standard letter-sized envelopes were licked. Half of the envelopes served as nonirradiated controls while the other half underwent irradiation at dosages sufficient to kill anthrax spores (29.3 and 51.6 kGy). Total cellular DNA was extracted from all envelopes; nuclear short tandem repeat loci, as well as the hypervariable region I from mitochondrial DNA, were amplified by means of the polymerase chain reaction. Short tandem repeat profiles and mitochondrial DNA sequence haplotypes were acquired on an ABI Prism® 310 Genetic Analyzer platform. Analysis of data from irradiated samples revealed evidence of DNA degradation; however, the ability to construct full genetic profiles from both nuclear and mitochondrial DNA remained largely unaffected. The use of the polymerase chain reaction, coupled with florescent fragment analysis and mitochondrial DNA sequencing, should be considered to profile biological material from evidence enduring irradiation to inactivate infectious agents.

DNA damage reduces Taq DNA polymerase fidelity and PCR amplification efficiency

“…PCR products were sized by agarose gel electrophoresis (data not shown) and purified using the Qiagen ® QiaQuick PCR amplification kit [23]. To prevent chemical degradation postpurification, purified products were eluted in Qiagen Buffer EB (10mM Tris-Cl, pH 8.5) and stored at −20° C.…”

“…Purified PCR products were sequenced using ABI Big Dye Terminator chemistry with Taq FS polymerase [23]. Due to the shortness of templates (< 100 bp), modifications to the reference protocol were made.…”

“…Sequencing reactions were purified using Centri-Sep 100 spin columns (Princeton Separations, Princeton, NJ; [23]). Samples were denatured at 95° C for 4 min and loaded onto an Applied Biosystems 310 Genetic Analyzer for analysis.…”

Modification of a human DNA extraction protocol to mitigate operator risk when analyzing human DNA evidence contaminated with risk group 3 bacteria

“…DNA analysis was performed to determine if there was any PCR interference or (Withrow et. al., 2003).…”

Comparison of the effects of sterilisation techniques on subsequent DNA profiling

“…In some instances, gamma radiation was shown to eliminate small amounts of genomic and plasmid DNA [4]. Withrow et al [5] demonstrated that full mitochondrial and nuclear profiles were obtained after beta radiation doses of 29.3 and 51.6 kGy even though it was demonstrated that the radiation significantly reduced the total amount of DNA extracted and that some evidence of degradation was present. Work conducted by Castle et al [6] corroborated this result and showed that the amount of DNA available for profiling is reduced after beta radiation but that 16 STRs (including all 13 CODIS STRs) were detectable after treatment.…”