Microarray-Based Detection of Bacteria by On-Chip PCR
4. Notes
5. This allows assignment of final results according to the spotting layout (see Note 15).
checked and, if required, cleaned using a precision cleaner such as the anionic detergent Alconox.
5. In our initial studies we screened a multitude of commercially available microarray glass slides and found that only slides containing a polymer-type surface coating in addition to the silane layer were suitable for on-chip PCR. The polymer layer increases the spatial distance between the glass-surface and the PCR primers, which decreases steric hindrance normally observed with DNA microarray sur- faces (28) and enhances interaction between primers, template DNA, and poly- merase at the primer binding region (13,29). Furthermore, we found that the attachment chemistry creating stable, covalent amide bonds between the polymer and the 5´ amino group of PCR primers is particularly compatible with the high temperatures during PCR. Thus, if combined with EGS crosslinker chemistry, other microarray glass slides containing a polymer-type surface coating are suit- able for on-chip PCR, such as dendrimer coatings (30,31) or the commercially available CodeLink Activated Slides (Amersham Biosciences).
6. We observed that the concentration of the oligos to be spotted could range between 15 and 25 µM. Although lower concentrations typically yield lower binding effi- ciencies, higher spotting concentrations also can result in lower efficiencies as a result of the electrostatic blockage of target hybridization at high surface probe density (27). Adjustment of spotting concentrations, however, might be required if other slide surfaces and/or other spotting technologies are used.
7. In addition to the Affymetrix spotter, which is a contact printer based on the so-called pin-and-ring technology, we also used a spotting robot that employs the widely used split-pin technology (TeleChem SMP 3 Stealth Pins). We advise one to carefully adjust spotting buffer, additives influencing surface tension such as SDS or DMSO, and spotting conditions like temperature and humidity according to the type of spot- ting technology used. Manufacturers of microarraying machines and substrates typi- cally provide protocols for an optimal operating range regarding the aforementioned parameters.
8. The microarray layout should be designed carefully to allow simultaneous testing with several allele-specific primers. It is wise to array at least three replicates across several identical subarrays for each solid-phase oligonucleotide. Also, include guide dots that contain, for instance, a biotinylated oligonucleotide and can serve as controls for spotting, coupling, and staining (see Fig. 1C). Ideally, the subarrays are spotted with physically different pins of the spotting robot to control for poten- tial differences and variations in deposited material or spot morphology. A “high- replicate” array pattern also provides auxiliary security in the event of possible technical troubles, such as scratches on the slide surface introduced during the handling of the slides. Moreover, we have demonstrated that replicates greatly facilitate downstream automated analysis steps in on-chip PCR like grid place- ment and spot finding (16).
9. Whenever possible, spotted slides should be stored at least 2 wk if the full bind- ing capacity of the slide surface is needed. It is wise to produce the slides in batches of 40 to 80 pieces because the spotted slides can be stored at least 5 mo without loss of activity.
10. The technical challenge lies in performing a PCR in a small volume that is spread in a very thin layer over an area of 22 ⫻ 22 mm between the glass slide and the glass cover slip. For satisfactory efficiency, the enzymatic reaction performed on a flat glass surface requires a number of additives to the reaction mixture. First, the reaction is performed under higher salt (300 mM KCl, 50 mM Tris) and magnesium concentration (3 mM) compared to conventional PCR and contains bovine serum albumin as a blocking agent. Second, the reaction contains a self-seal reagent, which polymerizes upon contact with air at high temperature and thereby seals the reac- tion mixture at the edges to prevent evaporation during PCR. It is important to stick to this protocol and to check for the proper magnesium concentration that might require adjustment when using high primer, template or dNTP concentration. Vir- tually a single pipetting step is needed to launch the reaction from a complete mas- ter mix, which represents an important benefit with respect to potential sample contamination risk inherent to all PCR applications. A complete master mix can be prepared together with the polymerase and self-seal reagent in larger batches and stored in aliquots in lyophilized form after the addition of trehalose as stabilizer agent (32,33). After that, the reaction can be started by simply adding the DNA sample diluted to a 13 µL volume.
11. We have not quantified the actual amount of template DNA necessary for the on-chip PCR, but we normally obtain excellent results with template DNA extracted, for example, from overnight cultures of clinical swab samples using the following procedure. 750 µL of an overnight culture is centrifuged at 10,000g for 5 min and the supernatant is discarded. The pellet is then resus- pended in 750 µL of PBS and centrifuged again. This second centrifugation and washing step yields better results, most probably as a result of the removal of possible PCR inhibitors (15). The pellet is finally resuspended in 200 µL of PBS and used for the DNA extraction procedure with commercial DNA extrac- tion kits. It is important to note that template quality and quantity requirements in on-chip PCR are very similar to that in conventional tube PCR. It is advis- able always to include positive control nucleic acid, such as DNA isolated from cultured reference strains, and negative control samples.
12. It is important that the cover slip that is used for sealing the reaction is very clean.
Either specially treated cover slips for microarray applications should be used (Amersham Biosciences) or standard cover slips should be cleaned by ultrasonic treatment in detergent. Alternatively, the reaction could be performed under frame seals that are offered by some microarray vendors (MJ Research). Such self-adhesive frames might be beneficial under certain circumstances, although the reaction volume needed rises to a 25 µL minimum.
13. We previously found that denaturing washing steps after the amplification proce- dure is not necessary, suggesting that no significant nonspecific hybridization of unbound PCR products to un-reacted solid-phase primers occurs (13). Under cer- tain circumstances, however, further stringent washing steps at high temperature could be useful to increase specificity.
14. Other dyes could be used, as well as the direct incorporation of commonly used Cy3 or Cy5-modified dCTP nucleotides. However, we have sometimes observed
higher background, most probably because of the denaturing effects on the glass surface of the fluorescence labels during thermal cycling. It should also be known that Cy5 dyes tend to degrade under ozone exposure (34).
15. Fluorescence intensities usually are calculated as spot medians after local back- ground subtraction. Data analysis and interpretation can be substantially facili- tated and automated by using scripting extensions. The Genepix software allows scripting extensions that can be used for automatically calculating discrimination scores from average values derived from the replicate spots. Automated routines for genotype assignment for bi-allelic variants have been described recently (17).
The method is of course compatible with any other commercially available microarray slide scanner and accompanying data analysis software.
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From: Methods in Molecular Biology, vol. 345: Diagnostic Bacteriology Protocols, Second Edition Edited by: L. O‘Connor © Humana Press Inc., Totowa, NJ