Comparison of DNA extraction kits for PCR-DGGE analysis of human intestinal microbial communities from fecal specimens

Abstract

Background: The influence of diet on intestinal microflora has been investigated mainly using conventional microbiological approaches. Although these studies have advanced knowledge on human intestinal microflora, it is imperative that new methods are applied to facilitate scientific progress. Culture-independent molecular fingerprinting method of Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis (PCR-DGGE) has been used to study microbial communities in a variety of environmental samples. However, these protocols must be optimized prior to their application in order to enhance the quality and accuracy of downstream analyses. In this study, the relative efficacy of four commercial DNA extraction kits (Mobio Ultra Clean(R) Fecal DNA Isolation Kit, M; QIAamp DNA Stool Mini Kit, Q; FastDNA SPIN Kit, FSp; FastDNA SPIN Kit for Soil, FSo) were evaluated. Further, PCR-DGGE technique was also assessed for its feasibility in detecting differences in human intestinal bacterial fingerprint profiles.

Method: Total DNA was extracted from varying weights of human fecal specimens using four different kits, followed by PCR amplification of bacterial 16S rRNA genes, and DGGE separation of the amplicons.

Results: Regardless of kit, maximum DNA yield was obtained using 10 to 50 mg (wet wt) of fecal specimens and similar DGGE profiles were obtained. However, kits FSp and FSo extracted significantly larger amounts of DNA per g dry fecal specimens and produced more bands on their DGGE profiles than kits M and Q due to their use of bead-containing lysing matrix and vigorous shaking step. DGGE of 16S rRNA gene PCR products was suitable for capturing the profiles of human intestinal microbial community and enabled rapid comparative assessment of inter- and intra-subject differences.

Conclusion: We conclude that extraction kits that incorporated bead-containing lysing matrix and vigorous shaking produced high quality DNA from human fecal specimens (10 to 50 mg, wet wt) that can be resolved as bacterial community fingerprints using PCR-DGGE technique. Subsequently, PCR-DGGE technique can be applied for studying variations in human intestinal microbial communities.

Figure 1

Average DNA yield obtained using the four commercial kits as influenced by fecal specimen weights. DNA was extracted from 200, 100, 50, 25, and 10 mg of human fecal specimens (n = 45/kit), using Mobio Ultra Clean^® Fecal DNA Isolation Kit (M), QIAamp^® DNA Stool Mini Kit (Q), FastDNA^® SPIN Kit (FSp), and FastDNA^® SPIN Kit for Soil (FSo). Values for DNA yield were normalized based on the dry weight of the respective fecal specimen. Means with different letter designation are significantly different (comparisons within each extraction kit; P < 0.05).

Figure 2

Quality of DNA extracted from varying weights of fecal specimens using kit FSo. DNA was extracted from 200, 100, 50, 25, and 10 mg of human fecal specimens using FastDNA^® SPIN Kit for Soil (FSo). Note shearing of DNA in extractions using higher fecal weights.

Figure 3

Average DNA yield obtained using the four commercial kits as influenced by fecal dry matter. The percent dry matter in the human fecal specimens were 26%, 35%, and 41% (n = 45/kit). Values for DNA yield were normalized based on the dry weight of the respective fecal sample. DNA from the fecal specimens were extracted using Mobio Ultra Clean^® Fecal DNA Isolation Kit (M), QIAamp^® DNA Stool Mini Kit (Q), FastDNA^® SPIN Kit (FSp), and FastDNA^® SPIN Kit for Soil (FSo).

Figure 4

Average DNA yield obtained using kits M and Q. Comparison was made on the average DNA yield of these kits with and without the addition of vigorous mixing using the FastPrep^® Instrument (n = 3/kit; M, Mobio Ultra Clean^® Fecal DNA Isolation Kit; Q, QIAamp^® DNA Stool Mini Kit). Values for DNA yield were normalized based on the dry weight of the respective fecal sample. Means with different letter designation are significantly different (P < 0.05).

Figure 5

A comparison of DGGE profiles of PCR amplified bacterial 16S rRNA gene. DNA was extracted using FastDNA^® SPIN Kit for Soil (FSo) using 25 mg of fecal specimens collected from four human subjects (Subject A, B, C, and D; n = 4 for each subject). Bacterial fingerprint profile is based on 35 to 60% DGGE gel gradient. Lane 1 to 4 show bacterial fingerprint profile of consecutive fecal samples collected from each subject.

Figure 6

Dendrograms generated from PCR-DGGE profiles obtained from DNA extracted using the four commercial kits. The dendrograms were based on Unweighted Pair Group Method with Arithmetic Averages (UPGMA). Dendrogram A is based on 35 to 50% DGGE gradient gel and dendrogram B is based on 45 to 60% DGGE gradient gel.