Subjects
Healthy volunteers aged 20 - 30 yr (n = 4) were recruited from a college community. Volunteers were non-smokers, did not have any food allergies, had not used antibiotics for the past 6 months, and did not have any history of GI diseases (stomach ulcers, colon cancer, recent bouts of diarrhea, acid reflux disease, heartburn). Female participants were not pregnant or lactating at the time of study. Protocols were approved by the Committee on the Use of Human Research Subjects at Purdue University, West Lafayette, IN.
Fecal Collection and Fecal Dry Weight Determination
Fecal specimens were collected from each volunteer once a month for a total of 4 specimens per volunteer, which were then stored at -20°C prior to being analyzed. To determine fecal moisture content, frozen specimens were thawed at 4°C. Then, approximately 0.5 g (wet wt) of each fecal specimen was placed in a vacuum dryer for 3 d and re-weighed. Percent fecal dry weight was calculated using the following formula:
To ensure sample homogeneity, remaining fecal specimens were diluted with sterile water (1:2 wt/vol) and then kneaded in separate sterile plastic bags using a stomacher at high speed for approximately 5 min. A sub-sample was aliquoted for DNA extraction and the remainder stored at -20°C.
DNA Extraction and Quantification
The following four commercial DNA extraction kits were evaluated:
M - Mobio Ultra Clean® Fecal DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA)
Q - QIAamp® DNA Stool Mini Kit (Qiagen Inc., Valencia, CA)
FSp - FastDNA® SPIN Kit (MP Biomedicals, Irvine, CA)
FSo - FastDNA® SPIN Kit for Soil (MP Biomedicals, Irvine, CA)
Three variables were tested for kit extraction efficacy: i) ratio of water to dry matter content of fecal specimens, ii) wet fecal specimen weight used for extraction, and iii) cell lysis method. Fecal specimens submitted by the subjects varied in their percent dry matter content, which may be correlated with the microbial concentration and subsequently the quantity of DNA extracted from the fecal specimens. This would make it difficult to use a standardized method in clinical studies. To investigate this, three fecal specimens with differing dry matter content (26%, 35%, and 41%) from different individuals were selected for extraction.
Protocols supplied with the kits recommended different amounts of starting materials for extraction (Table
1). Preliminary experiments showed that extracting from fecal specimens above 200 mg (i.e., 300 mg and 500 mg) were not feasible. This amount overloaded the purification matrix and caused the filter to rupture. Thus, five specimen weights (10, 25, 50, 100, and 200 mg (wet wt)) were selected to evaluate the efficacy of the DNA extraction kits.
Table 1
Comparison of recommended DNA extraction protocols based on technical booklets included with respective extraction kits.
Fecal wt (mg)
| 250 - 1000 | 180 - 220 | 200 | 500 |
Beads
| Unknown beads | None | Ceramic + garnet | Ceramic + silica |
Cell lysis and homogenization
| Flat bed vortexer (10 min) | Centrifuge (14,000 rpm, 1 min) | Fast Prep® Instrument (speed 6.0, 40 s) | Fast Prep® Instrument (speed 5.5; 30 s) |
Adsorption of inhibitors
| IRS
2
solution | InhibitEx tablet | None listed | None listed |
Approximate time to completion
3
| 45 to 60 min | 60 to 80 min | 45 to 60 min | 60 to 80 min |
Average cost of kit
4
| $176.00 | $181.00 | $344.20 | $240.45 |
Additional experiments with modifications to the standard protocol were conducted to determine whether the use of vigorous shaking, specifically using the FastPrep® Instrument, was the key determinant in influencing DNA yield. The two kits, M and Q, that did not use a FastPrep® Instrument, were tested by homogenizing 25 mg of a fecal specimen (26.3% dry matter) in their respective lysing matrices using the FastPrep® Instrument for 30 seconds at a speed setting of 5.5. The lysis solution Q did not contain any beads and none were added. Supernatant from each mixture was then processed using subsequent steps in the respective protocols of kits M and Q.
DNA yield was quantified by fluorometric analysis (Picofluor, Turner BioSystems, Sunnyvale, CA) using calf thymus DNA as a standard. Values for DNA yield were normalized based on the dry weight of the respective fecal specimen. DNA quality was evaluated using gel electrophoresis on 0.8% agarose gels stained with ethidium bromide, visualized on a UV transilluminator and photographed (UVP BioImaging system, UVP LLC, Upland, CA).
PCR-DGGE Analysis
PCR-DGGE technique was used to evaluate the microbial community profiles from the respective fecal specimens. Bacterial 16S rRNA gene V3 region was amplified by PCR using primers PRBA338F (5'
CGC CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GAC TCC TAC GGG AGG CAG CAG 3'; GC-clamp is in boldface) [
19] and PRUN518R (5' ATTA CCG CGG CTG CTGG 3') [
20]. The GC-clamp, which is a sequence that is rich in guanine and cytosine, is added to the 5' end of the forward or reverse primer in order to prevent DNA from being completely denatured into single strands. Subsequently, this improves band resolution in denaturing gels. The final PCR reaction mixture (50 μl total volume) contained 5 μl of 10× PCR Buffer, 4 μl of 25 mM MgCl
2, 0.4 μl of 100 mM deoxynucleotide triphosphate mixture, 2.5 μl of 20 mg/ml bovine serum albumin, 0.75 μl of each primer (at 25 μM), 1 μl of 5 U/μl Taq polymerase, and 1 μl DNA template (approximately 10 ng/μl). The amplification condition was 94°C for 5 min (initial denaturation), followed by 30 cycles of denaturation at 92°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 30 sec. A final extension step was carried out at 72°C for 15 min. Presence of PCR products were confirmed by electrophoresis on 1.5% agarose gels stained with ethidium bromide in 1× TAE buffer using Lambda DNA-Hind III Digest (New England BioLabs, Inc., Ipswich, MA) as a molecular weight standard. Gels were visualized on a UV transilluminator and photographed (UVP BioImaging system, UVP LLC, Upland, CA).
PCR amplicons were separated using DGGE, which was conducted using the DCode™ Universal Mutation Detection System (Bio-Rad Laboratories, Hercules, CA), with slight modifications to the method previously described by Muyzer et al. [
21]. Equal masses of PCR products were separated on 8% (wt/vol) polyacrylamide gels (40% acrylamide/bis solution, 37.5:1; Bio-Rad Laboratories, Hercules, CA) in 1× TAE (40 mM Tris, 20 mM Acetate, 1.0 mM Na
2-EDTA) using denaturing gradient ranges of 35 to 50%, 45 to 60%, and 35 to 60%, where a 100% denaturant contained 7 M urea and 40% (vol/vol) deionized formamide. Electrophoresis was performed at 50 V for 10 min, then at 200 V for 5.5 hr. Electrophoresis buffer (1× TAE) was maintained throughout at 60°C. Gels were then stained using SYBR Green I nucleic acid stain (Cambrex Bio Science, Rockland, ME), visualized on a UV transilluminator, and photographed (UVP BioImaging system, UVP LLC, Upland, CA).
Analysis of Bacterial DGGE Banding Profiles and Sequencing
Similarities between banding patterns in the DGGE profile were calculated based on the presence and absence of bands and expressed as a similarity coefficient. In this study, the Dice similarity coefficient was used to calculate pairwise comparisons of the DGGE fingerprint profiles obtained from the four DNA extraction kits. This similarity coefficient is calculated based on the following formula: D
sc = [2
j/(
a+
b)], where
a = number of DGGE bands in lane 1,
b = number of DGGE bands in lane 2, and
j = number of common DGGE bands in lane 1 and lane 2, and D
sc = 1 indicates identical profiles [
22]. Dendrograms showing clustering according to the similarity of banding patterns between samples were constructed by the unweighted pair group method of arithmetic averages (UPGMA) [
23] using BioNumerics software (BioNumerics, Applied Maths, Inc., Austin, TX).