Reliable Noninvasive Genotyping: Fantasy or Reality?

Abstract

Noninvasive genotyping has not gained wide application, due to the notion that it is unreliable, and also because remedial measures are time consuming and expensive. Of the wide variety of noninvasive DNA sources, dung is the most universal and most widely used in studies. We have developed collection, extraction, and amplification protocols that are inexpensive and provide a high level of success in amplifying both mitochondrial and nuclear DNA from dung. Here we demonstrate the reliability of genotyping from elephant dung using these protocols by comparing results from dung-extracted DNA to results from blood-extracted DNA. The level of error from dung extractions was only slightly higher than from blood extractions, and conducting two extractions from each sample and a single amplification from each extraction was sufficient to eliminate error. Di-, tri-, and tetranucleotide loci were equally reliable, and low DNA quantity and quality and PCR inhibitors were not a major problem in genotyping from dung. We discuss the possible causes of error in genotyping with particular reference to noninvasive samples and suggest methods of reducing such error.

Sex Determination of Sika Deer (Cervus nippon yesoensis) Using Nested PCR from Feces Collected in the Field

“…In this situation, fecal samples that can be collected noninvasively are simply counted instead of biomaterials and studies using fecal samples have been conducted in many species [2,5,11,23,25,30]. To use fecal samples for further analysis beyond genotyping, sex information will be essential.…”

Blood meal analysis of tabanid fly after it biting the rare Sumatran rhinoceros

“…Peer reviewer with the advent of polymerase chain reaction (PCR)-based technology [1][2][3][4][5] . This advancement had truly escalated the field of conservation biology particularly when dealing with rare and elusive, endangered, or cryptic fauna [1][2][3][5][6][7][8] .…”

“…This advancement had truly escalated the field of conservation biology particularly when dealing with rare and elusive, endangered, or cryptic fauna [1][2][3][5][6][7][8] .…”

Species identification, molecular sexing and genotyping using non‐invasive approaches in two wild bovids species: Bos gaurus and Bos javanicus

“…Unfortunately, these kinds of data are not easily obtained as the quality and quantity of DNA extracted from non-invasive sources (such as hair or dung) are generally poor. In addition, the risk of DNA contamination, allelic drop-out or scoring of false alleles is high [Taberlet et al, 1996;Fernando et al, 2003;Bonin et al, 2004]. This risk can be minimized through considerable repetition of extractions and polymerase chain reaction amplifications, and so high number of replicates are necessary to assess genotypes with certainty [Taberlet et al, 1996; but see Miller et al, 2002 for an alternative approach using maximum likelihood].…”

“…In the case of herbivores, the use of fecal DNA poses further problems owing to the inhibitory effects that some plant secondary compounds exert on PCR polymerases. As such, the success of PCR amplifications is highly dependent on the maximization of intact target DNA yield with the simultaneous minimization of inhibitors [Fernando et al, 2003]. In the particular case of ruminant species with compact feces pellets, the DNA extraction is easier than DNA extracted from nonpelleted feces because it can be optimized by using mucosal intestinal cells from the pellets' surface [Flagstad et al, 1999].…”