Forensic animal DNA analysis using economical two-step direct PCR

Abstract

Wildlife forensic DNA analysis by amplification of a mitochondrial locus followed by DNA sequencing is routine, yet suffers from being costly and time-consuming. To address these disadvantages we report on a low-cost two-step direct PCR assay to efficiently analyze 12 forensically relevant mammalian sample types without DNA extraction. A cytochrome oxidase I degenerate-universal primer pair was designed and validated for the developed assay. The 12 sample types, which included bone, horn, feces, and urine, were amplified successfully by the assay using a pre-direct PCR dilution protocol. The average amplification success rate was as high as 92.5 % (n = 350), with an average PCR product concentration of 220.71 ± 180.84 ng/μL. Differences in amplification success rate and PCR product quantity between sample types were observed; however, most samples provided high quality sequences, permitting a 100 % nucleotide similarity to their respective species via BLAST database queries. The combination of PBS and Phire(®) Hot Start II DNA polymerase gave comparable amplification success rate and amplicon quantity with the proprietary commercial kits (P > 0.05, n = 350) but at considerably lower cost. The stability of the assay was tested by successfully amplifying samples that had been stored for up to 12 months. Our data indicate that this low-cost two-step direct amplification assay has the potential to be a valuable tool for the forensic DNA community.

Ivory species identification using electrophoresis‐based techniques

“…The success could be due to several factors such as heating the samples, the use of dilution buffers (i.e. PBS), inhibitor‐resistant DNA polymerase, and PCR buffer; these helps to lyse cells, release DNA from ivory, all of which minimize inhibition of the PCR . Only a very small amount of ivory powder is required to obtain a successful amplification (such as a tiny scraping that sits under the 20 μL PBS), being beneficial for processed or degraded ivory and also tiny invasive samples .…”

“…Evaluation of the possibility of using direct amplification was tested using 20 confiscated ivory samples were analyzed with the developed assay using a conventional DNA extraction and direct amplification following protocol of Kitpipit et al. .…”

Evaluation of poly(ethylene glycol)-coated monodispersed magnetic poly(2-hydroxyethyl methacrylate) and poly(glycidyl methacrylate) microspheres by PCR

“…The PCR amplification of nucleic acids has become a powerful diagnostic tool to identify microorganisms in foods [30,31], clinical samples [32] and forensic analysis [33]. Because PCR amplifies the regions of the DNA that it targets, it can be used to analyze extremely small amounts of the samples [28].…”

The Evaluation of Magnetic Polymethacrylate-based Microspheres Used for Solid Phase DNA Micro-Extraction

“…Amplification of nucleic acids using polymerase chain reaction (PCR) has become a powerful diagnostic tool for the identification of microorganisms in foods [1][2][3][4], soil communities [5], clinical samples [6], forensic analysis [7], etc. Usually, there is no problem with the amplification of DNA isolated from pure bacterial cultures.…”