IC patients were chosen by following the criteria of the National Institute of Arthritis, Diabetes and Kidney Diseases (Gillenwater et al. 1987). Biopsies (approximately 1 to 5 mg) were obtained from bladders of IC patients and placed in a plain sterile cup with no solution. Biopsies were placed at -70°C within one hour after surgery (Human, L. personal communication). Total DNA was isolated from the biopsies by SDS lysis and followed by pronase and RNase treatment. Precipitated DNA was resuspended in water and quantified using absorbance at 260 nm (Domingue et al. 1995).

Bacterial 16S rRNA gene sequences were amplified by PCR using 16S rRNA universal and nested primers from extracted IC bladder biopsies DNA. PCR was performed as followed: 16S rRNA universal primers, 1 m g of 5’ primer 1A and 3’ primer 5 (Table 1A) , 5 units Thermus aquaticus DNA polymerase, 2 mM dNTPs and PCR buffer (10mM Tris-HCl, 50mM Kcl, 1 m g/ml gelatin and 2mM MgCl₂) were used in the first PCR reaction. Reactions were performed for 25 cycles using a BIOCYCLER thermal cycler (BIOS, New Haven, CT) programmed for 95° C denaturation, 58° C annealing and 72° C extension temperatures, with the first three cycles having extended denaturation and annealing times. After completion of the first PCR, 10% of reaction mixture was used for the second PCR using with the same conditions with the nested primers, 5’ primer 3 and 3’ primer 4 (Table 1A).

Among these clones, the blind control (MG), was not PCR amplified by these 16S rRNA primers. It was PCR amplified by primers, MgPa-1 and MgPa-3, which amplified the adhesion protein gene from pure culture DNA (Table 1B).

Two strategies could be used to sequence PCR-amplified 16S rRNA bacterial genes. One was to directly sequence the PCR products; the other approach involved cloning the PCR products and sequencing the plasmid-cloned inserts. In this thesis, these PCR products were cloned into the pUC18 vectors followed by sequencing. Cloning was utilized because the amount of IC patients’ biopsies was limited. Cloning would allow PCR products to be stored in vectors and avoids the necessity of taking multiple samples from the patient. Second, the PCR products could be heterogeneous. Cloning could preserve the variety to construct a library of these DNA domains. The PCR products were cloned into the pUC18 plasmid SmaI site by blunt-end ligation.

Cloned 16S rRNA genes were prepared for sequencing as follows: pUC18-IC DNA was extracted by QIAprep-spinÒ Plasmid Kit from E. coli according to the manufacturer’s direction (Qiagen, Chatsworth, CA). Cloned IC DNA sequences were amplified using pUC18 primers (Table 1C) that flanked the cloned insert. To duplicate sequencing reaction and confirm the fidelity of sequences, each clone was amplified by two different sets of primers (Fig. 1). One PCR reaction included 1 pmol biotinylated pUC18 forward primer (primer FB), a corresponding non-biotinylated reverse primer (primer R) and 70 picograms template DNA in 100 ul Taq polymerase reaction buffer (50 mM KCl, 10 mM Tris-HCl pH 8.8 ,0.1% Triton X-100) containing 2.5 mM MgCl2, 0.2 mM of each dNTP, and 2.5 units of Taq DNA polymerase (Promega, Madison, WI). Another PCR reaction included 1 pmol non-biotinylated pUC18 forward primer (primer F), a corresponding biotinylated reverse primer (primer BR). A drop of DNase/RNase free mineral oil (Sigma, Saint Louis, MO) was placed over each reaction to prevent evaporation. Thirty-five cycles of PCR were performed on a programmable thermal cycler (MJ Research Inc. Watertown, MA) using the following parameters: denaturation at 94° C for 30 seconds, annealing of primers at 55° C for 30 seconds and extension of synthesized strands at 72° C for 1 minute with a final cycle in which the extension time was extended to 2 minutes to facilitate strand extension. PCR products were purified by QIAquickÒ PCR Product Purification Kit (Qiagen, Chatsworth, CA) to remove unincorporated primers and free nucleotides according to the manufacturer’s direction. The purified PCR products were electrophoresed in 1% agarose gel (SeaKem LE, FMC Rockland, ME) to verify amount of DNA for analysis. Both of biotinylated and non-biotinylated primers were synthesized by the LSU Medical Center Core Laboratory (LSUMC New Orleans, LA).

For the preparation of single-stranded DNA as a sequencing template, streptavidin-linked to magnetic beads were used. Two to five micrograms of a biotinylated PCR product in 40 ul TE was added to 200 micrograms of streptavidin-coated paramagnetic beads (Dynabeads M280; DYNAL AS, Oslo, Norway) in a total volume of 80 ul for 10 minutes. Prior to adding PCR product, streptavidin-coated paramagnetic beads were prewashed twice with 2X binding and washing buffer (B&W buffer: 10 mM Tris-HCL pH 7.5, 1.0 mM EDTA, 2.0 M NaCl, 0.1% Tween-20). The biotinylated amplicons from the target DNA bound to the streptavidin-coated magnetic beads. The immobilized product was placed in a Dynal magnet for 10 minutes and the supernatant removed with a pipette. The beads were washed by adding 40 ul of 2X B&W buffer, collected on the side of the tube with the Dynal magnet and the supernatant was removed. Beads incubated in 8 ul of freshly prepared 0.1 M NaOH solution at room temperature for 10 minutes to denature double strand DNA (Fig. 1A). The beads with the immobilized biotinylated DNA were washed once with 50 ul 0.1 M NaOH, once with 40 ul B&W buffer, and three times with 50 ul TE buffer pH 7.5 (containing 1.0 M NaCl). The beads containing single strand template DNA were suspended in 9 ul water.

Sequencing was performed according to Sanger et al. (1977). The 9 ul solution of beads and template DNA was added to 7.5 ul Sequenase reaction mixture. Each clone was sequencing in two directions, so each clone had two sets of single-stranded DNA as sequencing template (Fig. 1A). Set FB single-stranded DNA contained biotinylated forward primer sequences; set RB single-stranded DNA contained biotinylated reverse primer sequences. Set FB was incubated with 5 ul 5 pM reverse primers and set RB with 5 ul 5 pM forward primer. The reaction mixture was heated to 100° C for 3 minutes and cooled on ice for annealing, then 8 ul of labeling mix (25 mM DTT, 10 uCi [³⁵S]dATP, dNTPs mixture and 9.4 units Sequenase 1.6 units/ul) (Sequenase PCR Product sequencing kit, USB Cleveland, Ohio ) was added. Total reaction was divided equally into four tubes each containing 2.5 ul dideoxy dNTPs which had been preincubated at 37° C for 5 minutes. The reactions were stopped by addition of 4 ul stop mix. Both FB and RB of each clone were analyzed by denaturing polyacrylamide gel electrophoresis (BRL Model 2, Gaitherburg Maryland) on 8M urea (Biorad, Hercules, CA) 6% polyacrylamide (USB, Cleveland, OH), respectively. After sequences were analyzed from FB and RB of each clone, each clone contained the sequence of 5’ region, the overlapped region and the 3’ region (Fig. 1B).

Sequences of all regions were organized and stored in PC/Gene (A. Bairoch/ University of Geneva/ Switzerland; IntelliGenetics Inc.). Phylogenetic analysis was determined using PC/Gene. The identity of DNA sequences was determined by comparison to two computer databases of known sequences (NCBI and RDP) through electronic-mail (e-mail) server. The program, the BLAST (Basic Local Alignment Search Tool), (Altschul et. al. 1990) was obtained from the network server (Blaster) at the National Center for Biotechnology Information (NCBI) of the NIH. The BLAST algorithm is a heuristic for finding upgapped, locally optimal sequence alignments. The NCBI includes the Brookhaven Protein Data Bank, the GenBank, the EMBL Data Library and the DDBJ (Databases of Japan).

The RDP (Ribosomal Database Project) at the University of Illinois compiles ribosomal sequences and related data and redistributes them in aligned and phylogenetically ordered form. These sequences are drawn from various rRNA collections such as the GenBank and other individual laboratories around the world. As of current release, this database contains over 3000 aligned small subunit (SSU) and large subunit (LSU) ribosomal RNA sequence. Prokaryotic sequences predominate.