Microarray-Based Detection of Bacteria by On-Chip PCR
3. Methods
7. Glass cover slips (22 ⫻ 22 mm), for microarray applications.
8. PTC 200 in situ slidecycler (MJ Research [Bio-Rad]).
9. Phosphate-buffered saline (PBS) buffer: 0.14 M NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4 (as required during DNA extraction).
2.5. Fluorescence Staining and Scanning
1. Standard saline citrate buffer: 150 mM sodium chloride, 15 mM sodium citrate, pH 7.0. Store as 20X stock solution.
2. 10% (w/v) SDS solution.
3. Streptavidin-Alexa Fluor 647 conjugate (Molecular Probes). Store under light protection according to the manufacturer’s instructions.
4. TBST buffer: 150 mM NaCl, 10 mM Tris-HCl, 0.5% Tween-20, pH 8.0.
5. Affymetrix 428 laser scanner (Affymetrix), or similar microarray scanner.
6. GenePix software (Axon Instruments), or any other software for microarray analysis.
3.1.2. Design of Species-Specific Solid-Phase Primers
1. For the development of bacterial-specific primers, all partial 23S rDNA sequences of the bacterial identification panel are aligned using the multiple sequence align- ment computer program GeneDoc.
2. The sequences are obtained either by prior sequencing or from public databases (e.g., the EMBL nucleotide sequence database, available from: http://www.ebi.ac.
uk/embl/).
3. The alignment is screened for nucleotide positions unique to specific species.
4. The solid-phase primers are designed such that the 3´ end interrogates the spe- cies-specific nucleotide positions. Include positive and negative control primers here. Positive controls interrogate conserved nucleotide positions and allow con- trol of the successful amplification with the universal primer set. Negative con- trols can be used for monitoring specificity.
5. The specificity of the primers is then tested against known bacterial sequences using the blastn algorithm (Available at http://www.ncbi.nlm.nih.gov/blast/).
6. The primers are designed with a length between 30 and 40 nucleotides and annealing temperatures between 68 and 72⬚C.
7. The primers are synthesized with a 5´ terminal (CH2)6-NH2 modification (see Note 3).
3.2. Coating of Glass Slides 3.2.1. Cleaning
1. The glass slides are cleaned by incubation in a solution containing HCl:methanol (1:1) for 24 h at room temperature (see Note 4).
2. The slides are then washed thoroughly with deionized water and dried with com- pressed air.
3.2.2. Silane Derivatization
1. Incubate the cleaned slides in a solution containing 3% (v/v) trimethoxysilyl- propyl modified polyethyleneimine (see Note 5) in 95% ethanol for 5 min with vigorous agitation using a magnetic stirring bar at room temperature. It is impor- tant that the silane is freshly diluted from a stock stored under argon protection.
2. Wash the slides in 95% ethanol to remove remaining silane and dry using com- pressed air.
3. Cure the silane layer by baking the slides in an oven at 80⬚C for 1 h.
3.2.3. Surface Activation
1. Treat the slides with EGS by pipetting 100 µL of a 100 mM solution in DMSO between two slides. It is important that the EGS is freshly prepared from a stock stored under argon protection.
2. Place the two-slide sandwich upon a strip of parafilm on the laboratory bench and incubate overnight at room temperature.
3. Wash the slides in glass trays containing 250 mL of water, dry using compressed air, and seal in plastic containers using either vacuum sealing or desiccation bags.
3.3. Attachment of Solid-Phase Primers
3.3.1. Dissolving Oligonucleotides for Spotting
1. Dissolve the primers in spotting buffer at a concentration of 20 µM (see Note 6).
2. Pipet 20 µL of DNA primer solution into either 96-well or 384-well microtiter plates depending on the spotting robot used.
3.3.2. Spotting of Glass Slides
1. Spot the slides using an Affymetrix 417 Arrayer equipped with 125 µm pins, which results in spots approx 200 µm diameter.
2. Before and during spotting, ambient temperature and air humidity must be con- trolled and maintained at 20⬚C and 50 to 60% relative humidity, respectively (see Note 7).
3. The spotting layout (see Note 8) is determined by the print head geometry and the well positions of specific DNA oligos within the plates. Each slide can easily hold two arrays, which require a 22 ⫻ 22-mm area for on-chip PCR.
3.3.3. Binding to Solid Support
1. Transfer the arrayed slides from the arraying robot into a humid chamber (NaCl- saturated) and incubate at room temperature for 16 h.
2. Transfer the glass slides directly into plastic containers and store at room tem- perature, either vacuum-sealed or using desiccation bags (see Note 9).
3.4. On-Chip PCR
3.4.1. Preparation of PCR Master Six
1. Prepare a master mix as follows. A 13-µL final volume for each reaction is used.
2X HotStarTaq PCR buffer
100 µM of dATP, dGTP, dCTP, and 65 µM dTTP 35 µM Biotin-16-dUTP
0.25 µg/µL bovine serum albumin 1 U HotStarTaq DNA polymerase
1.4 µM of each liquid-phase primer (see Note 10).
3.4.2. Blocking of Glass Slides
1. Immediately before use in on-chip PCR, immerse the glass slides in blocking buffer for 20 min at 55⬚C. This blocking step is very important because it neutral- izes any residual un-reacted amine-reactive EGS groups.
2. Wash the slides with deionized water and dry using compressed air.
3. Put the slide on a clean and flat surface, ideally above a printout template, which visually indicates the positions and areas covering the two oligonucleotide arrays.
3.4.3. Starting the Reaction
1. Add the required amount of Self-Seal Reagent (25% v/v) to the prepared master mix from Subheading 3.4.1. and mix.
2. Add a 13-µL aliquot to separate tubes containing the DNA template (see Note 11) and mix carefully using the micropipet.
3. Pipet the master mix onto the slide, directly down onto the oligonucleotide array.
4. Immediately seal the reaction droplet by mounting a cover slip (see Note 12) using plastic forceps. Take care not to trap air bubbles when sealing the reaction droplet underneath the cover slip. Always use gloves when handling slides and do not touch the slide on the upper, flat surface containing the spots.
3.4.4. Thermal Cycling
1. Transfer the glass slides into a PTC 200 slide thermocycler and carry out the PCR according to the following scheme:
80⬚C for 10 min, 95⬚C for 5 min
10 cycles at 95⬚C for 30 s, 66⬚C for 25 s, and 72⬚ C for 35 s, followed by 25 cycles at 95⬚C for 20 s, 66⬚C for 10 s, and 72⬚C for 20 s, with a final
extension step of 3 min at 72⬚C.
2. The cycling parameters could require adjustment and empirical optimization, depending on target sequence context and length.
3.5. Fluorescence Staining and Scanning 3.5.1. Fluorescence Dye Staining
1. After cycling place the slides in a glass tray filled with washing buffer containing 0.1X standard saline citrate supplemented with 0.1% SDS and incubate with gentle agitation until the cover slips swim off (takes approx 10 min).
2. Wash the slides again in fresh buffer as directed previously for 10 min, followed by a short rinse in deionized water (see Note 13).
3. Dry the slides using compressed air.
4. Stain the slides by pipetting a 20-µL droplet of staining solution containing 0.02 µg of Streptavidin-Alexa Fluor 647 conjugate in TBST buffer onto the array (see Note 14).
5. After a 2-min incubation at room temperature, wash the glass slides for 5 min in TBST buffer to remove excess dye. Rinse with deionized water and dry under an air stream.
3.5.2. Fluorescence Scanning
1. Scan the slides at 10-µm pixel resolution with excitation at a wavelength of 635 nm using an Affymetrix 428 laser scanner according to the manufacturer’s in- structions.
2. The scanner yields a 16-bit gray scale image that is saved for further data analysis.
3. Using the GenePix software, the fluorescence scan images are analyzed by plac- ing a grid onto the image and performing the required spot finding and fluores- cence intensity extraction steps.
4. After checking for inter-replicate consistency between the subarrays, a mean fluorescence value is calculated for each specific probe.
5. This allows assignment of final results according to the spotting layout (see Note 15).