SSH enrichment of desired sequences

Faecal samples used in this study were obtained from a subsequent trial in 2007. Samples

were collected from a set of 12 male domestic piglets (4 different litters with a shared sire)

obtained from a commercial piggery (Boen Boe piggery, Mittagong, NSW). Piglets were housed

and fed as described above (section 3.2.1.1). Samples were obtained from the piglets over a 3

week period, from 17 to 32 days of age. Samples were collected immediately following

defecation and stored at -20°C until analysis.

3.2.2.2

DNA extraction

1 g faecal sample was homogenised in 5 ml TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5)

before DNA extraction as outlined in the general materials and methods (section 2.2). The

yield and size range for the DNA extracts were assessed by agarose gel electrophoresis on 1%

agarose gels in 0.5X TBE run at 100 V for 20 mins (section 2.4.1).

3.2.2.3

Whole genome amplification

DNA samples were whole genome amplified using the REPLI-g® UltraFast Mini Kit (QIAGEN, USA). Denaturation buffer (Buffer D1) and neutralization buffer (Buffer N1) was freshly

prepared according to manufacturer’s instructions. 1 μl of template DNA was placed into a

microfuge tube and mixed with 1 μl Buffer D1 by vortexing. The mixture was centrifuged

briefly and incubated at 20°C for 3 mins. 2 μl of Buffer N1 was then added, mixed and

centrifuged as before. A master mix was prepared on ice containing 15 μl of REPLI-g UltraFast

Reaction Buffer and 1 μl of REPLI-g UltraFast DNA Polymerase per reaction. 16 μl of this was

added to each reaction and the tubes were incubated at 30°C for 1.5 hours. Enzyme

inactivation was carried out at 65°C for 3 mins.

To test the yield obtained, 1 μl of amplified product was run out alongside 0.5 μg of 1kb DNA

staining for 20 mins in ethidium bromide, destaining in dH2O for 10 mins and visualisation

under UV transillumination. The size range was also examined via gel electrophoresis by

running out 1 μl of amplified product alongside 0.5 μg of 1 kb DNA ladder on a 1% agarose gel

in 0.5X TBE buffer. 5 μl of FastPrep DNA extract was also loaded and the gel was run for 40

mins at 240 V before staining, destaining and visualising as before.

3.2.2.4

Suppression subtractive hybridisation (SSH)

The flowchart in Figure 3.4 details the suppression subtractive hybridisation (SSH) method

with steps labelled as described in section 2.5.

FIGURE 3.4 Flowchart detailing the suppression subtractive hybridisation (SSH) method. Steps

are labelled from section 2.5.1-2.5.4 corresponding to the methods detailed in section 2.5.

3.2.2.5

DGGE validation

To assess the outcome of SSH fractionation, DGGE community profiling was conducted. PCR

amplification and DGGE analysis was conducted as described in section 2.3.1 and 2.4.2.

profile. After silver staining (section 2.4.2.1), analysis was conducted in Quantity One (Bio-Rad

Laboratories, USA).

3.2.2.6

Cloning and sequencing

Bands of interest on the DGGE gel were excised and placed in separate tubes and labelled. 20

μl of Milli-Q water was added and the gel slices were crushed with a pipette tip to resuspend

the DNA. 4 μl of each resuspension was used in a PCR with primers 968-F-GC and R-1401 [265],

scaled up to a total volume of 50 µl, using the same cycling parameters as previously described

(section 2.3.1). A 0.5% agarose gel in 0.5X TBE was run at 180V for 20 mins to confirm

amplification success. The remaining PCR product was purified using the QIAquick® PCR Purification Kit (QIAGEN, USA) according to manufacturer’s instructions. The purified products

were examined by running out another gel.

These products were then cloned using the TOPO TA Cloning® Kit (Invitrogen Corporation, USA) according to manufacturer’s protocols. The transformed E. coli cells were spread-plated

on LB agar containing 25 μg/ml kanamycin and 40 μg/ml X-Gal to select for transformants and

to enable blue-white screening, respectively. The plates were incubated overnight at 37°C.

8 white colonies were picked from each band type and patched onto a new LB/kanamycin/X-

Gal plate. Colonies were also screened by PCR with primers M13F (5’- G TAA AAC GAC GGC

CAG T-3’) and M13R (5’- CAG GAA ACA GCT ATG AC - 3’). A 25 μl reaction volume consisting of

1X Thermopol buffer, 5 mM dNTPs, 15 pmoles each forward primer and reverse primer, and 1

U Taq DNA Polymerase was used. The same PCR program as used for primers 968-F-GC and R-

1401 was used. 5 μl of product was then mixed with loading buffer and run out on 1.5%

agarose in 0.5X TBE alongside 0.5 μg of 100 bp DNA ladder. The gel was run at 240 V for 25

mins, before staining for 20 mins in ethidium bromide, destaining for 10 mins in dH2O and

One successful colony was picked from each band type and used to inoculate a 1 ml volume of

LB broth containing 25 μg/ml kanamycin. The samples were shaken horizontally at 37°C

overnight. The overnight culture was then spun down at maximum speed for 1 minute to

pellet the cells. The plasmid DNA was then purified using the PlasmidMAX™ DNA Isolation Kit

(Epicentre Biotechnologies, USA) according to manufacturer’s instructions. 2 μl of purified

plasmid DNA was mixed with loading buffer and run out on a 1% agarose gel alongside 0.5 μg

of 100 bp DNA ladder. The gel was run in 0.5X TBE buffer at 180 V for 30 mins before staining,

destaining and imaging as before.

The purified plasmid DNA was then sent to the Australian Genome Research Facility

(Queensland, Australia) for sequencing along with 1 pmole of 1401R. The retrieved sequences

were then examined and edited in Vector NTI (Invitrogen Corporation, USA). The flanking

sequences including the primers were removed before performing a search with BLAST (Basic

Local Alignment and Search Tool) against the National Centre for Biotechnology Information

(NCBI) microbial genome database [301]. From the data obtained, relevant sequences were

aligned and a phylogenetic tree of 16S rRNA sequences was constructed via the neighbour-

joining method in the Ribosomal Database Project (RDP) Phyllip Interface [302].

3.2.3 Fosmid library generation

Using a representative sample (from section 3.2.2.1), a fosmid library was constructed using

the methods detailed in section 2.6. The titre of the packaged fosmid clones was determined

and the presence and variability of inserts in the clones were determined as described in

section 2.6.5. Packaged clones were stored in glycerol (20% final concentration) at -80°C for