Beijing Institute of Microbiology and Epidemiology, Beijing, China
Manufacture protocol
Based on the whole-genome sequences of Y. pestis CO92, KIM and 91001, a total number of 4015 annotated open reading frames (ORFs) were selected after the exclusion of ORFs encoding IS protein, integrase, and transposase (The word “gene” will be used in the following part of this paper in reference to the ORF). Those highly homologous to each other at nucleotide level were also excluded. Gene specific primer pairs (16bp to 18bp in length) were designed to amplify nearly the full length of each gene. All primers were synthesized by Illumina Co. (San Diego, CA, U. S. A). 300µl of PCR product was obtained for each gene. PCR amplifications were performed in 96-well plates by using a DNA thermocycler (Biometra UNOII). 100 µl of PCR reaction mixture, containing 50 mM KCl, 10 mM Tris-HCl (pH8.0), 2.5 mM MgCl2, 0.001% gelatin, 0.1% BSA, 150 µM of each dATP, dCTP, dGTP and dTTP, 0.6 µM of each primer, 5 units of Taq DNA polymerase (MBI Fermentas), and 20 ng of template DNA, was predenatured at 95°C for 5 min. Then 30 cycles of 94°C for 1 min, 60°C for 1 min 30 sec and 72°C for 2 min were performed with a final extension at 72°C for 10 min. Each PCR product was verified by agarose gel electrophoresis and was scored as successful if a single band of the expected mobility was detected. DNA smear, multiple bands, or single band of unexpected size were characterized as unsuccessful reactions. Primer pairs from unsuccessful PCR reactions were re-amplified with altered annealing temperatures to get the desired single band of products. A few primers had to be redesigned to ensure successful amplification. In the end, 4005 genes were amplified successfully without any byproduct from strain 91001 or 82009 (Y. pestis 82009, a biovar Orientalis strain isolated from a natural focus in China, was used as an alterative of CO92 in this study). PCR products were purified by using 96-well Multiscreen PCR plates (Millipore) following the user manual’s instruction. The purified DNAs were air dried at 65°C, and were resuspended in 30 µl of 50% DMSO solution. 10 µl of the purified PCR product was transferred into Genetix 7020 384-well Polystyrene Plates for microarray printing. Each of the PCR products was spotted in duplicate on CSS-1000 silylated glass slides (CEL) by using a SpotArray72 Microarray Printing System (Perkin Elmer Life Sciences) with 32 Telechem SMP3 Stealth Pins (4×8 Layout). Genomic DNA of strain 91001, salmon sperm DNA and 50% DMSO solution were included in the microarray design as internal control elements. The spotted slides were stored in dry environment at room temperature for at least 12 hr, and then were crosslinked twice by using a UV Stratalinker (Hoefer) each at a total energy of 60 microjoules. The spotted CSS-1000 silylated slides need not any blocking procedure, such as the blocking of unreacted aldehyde groups and the prehybridization. The microarray slide to be hybridized was simply washed in water for 2 min to remove the unbounded DNA and then was blown to dry with hot air.
