Comparison of DNA extraction methods for polymerase chain reaction amplification of guanaco (Lama guanicoe) fecal DNA samples

Abstract

ABSTRACT. Feces-based population genetic studies have become increasingly popular. However, polymerase chain reaction (PCR) amplification rates from fecal material vary depending on the species, populations, loci, and extraction protocols. Here, we assessed the PCR amplification success of three microsatellite markers and a segment of the mitochondrial control region of DNA extracted from field-collected feces of guanaco (Lama guanicoe) using two protocols -Qiagen DNA Stool Kit and 2 cetyltrimethylammonium bromide/ phenol:chloroform:isoamyl alcohol (2CTAB/PCI) method. Chelex resin treatment to remove inhibitors was also tested. Our results show that the mitochondrial locus was the most difficult to amplify. PCR success rates improved for all markers after Chelex treatment of extracted DNA, and 2CTAB/PCI method (95.83%) appeared to perform slightly better than PCR amplification from guanaco fecal DNA samples stool kit (91.67%) for the nuclear markers. Amplification success was significantly influenced by the extraction method, Chelex treatment, and locus (P < 0.001) but not by the freshness of the feces (fresh vs old, P = 0.17). The repeatability levels were high without Chelex treatment (> 0.89), but they decreased slightly after treatment for amplification of nuclear markers and markedly after treatment for amplification of the mitochondrial control region. Thus, we showed that Chelex treatment gives high PCR success, especially for nuclear markers, and adequate DNA extraction rates can be achieved from L. guanicoe feces even from non-fresh fecal material. Although not significant, 2CTAB/PCI method tended to provide higher successful amplification rates on a whole set of samples, suggesting that the method could be particularly useful when using small sample sizes.

Genetic diversity of the pampas deer (Ozotoceros bezoarticus) population in the Brazilian Pantanal assessed by combining fresh fecal DNA analysis and a set of heterologous microsatellite loci

“…These results indicate that the use of fresh fecal samples can enhance the success of studies using fecal DNA samples ( Oliveira and Duarte, 2013 ). In contrast, based on samples collected in northern Chile during the summer, Espinosa et al (2015) concluded that fresh feces (based on the presence of mucus or deer observed defecating) and non-fresh feces (no mucus, deer not observed defecating) were equally efficient for successful DNA amplification in ungulate species.…”

The impact of different DNA extraction methods on the analysis of microbial diversity of oral saliva from healthy youths by polymerase chain reaction-denaturing gradient gel electrophoresis

“…The cause could be expressed in two aspects. Firstly, the abundance of the microbiota in the initial samples might have changed after DNA extraction 11 . This change might be relevant to the efficiency of the lysis buffer and the sensitivity of various microbiota to the lysis buffer from different extraction methods.…”

Fecal DNA isolation and degradation in clam Cyclina sinensis: noninvasive DNA isolation for conservation and genetic assessment

“…In addition, with the development of molecular biology technology, fecal DNA is extensively used in genetic biology studies for species identification [15–17], individual identification [18–20], sex identification [21–25], population genetic structure [26–28], and genetic diversity evaluation [29]. However, fecal sampling has some problems, such as poor fecal DNA isolation quality and low success rate of PCR amplification [30]. Moreover, no study has performed fecal DNA extraction on invertebrates, especially shellfish.…”