A comparison of the efficiency of five different commercial DNA extraction kits for extraction of DNA from faecal samples

Shantelle Claassen,Elloise Du Toit,Mamadou Kaba,Clinton Moodley,Heather J Zar,Mark P Nicol

doi:10.1016/j.mimet.2013.05.008

Shantelle Claassen, Elloise Du Toit + Show 4 more

Open Access

https://doi.org/10.1016/j.mimet.2013.05.008

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Abstract

Differences in the composition of the gut microbiota have been associated with a range of diseases using culture-independent methods. Reliable extraction of nucleic acid is a key step in identifying the composition of the faecal microbiota. Five widely used commercial deoxyribonucleic acid (DNA) extraction kits (QIAsymphony® Virus/Bacteria Midi Kit (kit QS), ZR Fecal DNA MiniPrep™ (kit Z), QIAamp® DNA Stool Mini Kit (kit QA), Ultraclean® Fecal DNA Isolation Kit (kit U) and PowerSoil® DNA Isolation Kit (kit P)) were evaluated, using human faecal samples. Yield, purity and integrity of total genomic DNA were compared spectrophotometrically and using gel electrophoresis. Three bacteria, commonly found in human faeces were quantified using real time polymerase chain reaction (qPCR) and total bacterial diversity was studied using denaturing gradient gel electrophoresis (DGGE) as well as terminal restriction fragment length polymorphism (T-RFLP). The measurements of DNA yield and purity exhibited variations between the five kits tested in this study. Automated kit QS exhibited the best quality and highest quantity of DNA. All kits were shown to be reproducible with CV values≤0.46 for DNA extraction. qPCR results showed that all kits were uniformly efficient for extracting DNA from the selected target bacteria. DGGE and T-RFLP produced the highest diversity scores for DNA extracted using kit Z (H′=2.30 and 1.27) and kit QS (H′=2.16 and 0.94), which also extracted the highest DNA yields compared to the other kits assessed.

Highlights

Variations in the composition of the gut microbiota between individuals are increasingly recognized due to the development of cultureindependent analytic techniques (Smith et al, 2011)
Evaluation of genomic Deoxyribonucleic acid (DNA) extracted from faecal samples before and after RNase treatment
Even though no difference was observed for kit QA with regard to DNA yield after RNase treatment, there was a significant change in the DNA yield following an RNA clean-up step (Fig. 1A), for both 100 mg (1294 ng; p b 0.0001) and 200 mg (2128 ng; p b 0.0001) of faecal material

Summary

Introduction

Variations in the composition of the gut microbiota between individuals are increasingly recognized due to the development of cultureindependent analytic techniques (Smith et al, 2011). Extracted genomic DNA needs to represent all microbial communities present within the sample in order to effectively characterize the microbiota present (Yuan et al, 2012). This poses further challenges as the human gastro-intestinal tract (GIT) is

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