Microarray-based detection of bacteria by on-chip PCR.
In this chapter, a protocol called on-chip polymerase chain reaction (PCR) is presented for the deoxyribonucleic acid (DNA) microarray-based detection of bacterial target sequences. On-chip PCR combines, in a single step, the conventional amplification of a target with a simultaneous, nested PCR round intended for target detection. While freely diffusible primers are deployed for amplification, the nested PCR is initiated by oligonucleotide primers bound to a solid phase. Thus, on-chip PCR allows the single-step amplification and characterization of a DNA sample as a result of separation in liquid and solid-phase reactions. In contrast to conventional PCR, the reaction is performed directly on the flat surface of a glass slide that holds an array of covalently attached nested primers. The bacterial target DNA is amplified and probed using primers identifying either species-specific sequence regions of ribosomal DNA or unique bacterial target genes, such as virulence or resistance factors. The microarray is produced using standard spotting equipment with an array layout containing a high number of replicates. Fluorescence scanning of on-chip PCR slides allows the rapid detection of the target of interest. The protocol described herein will show how on-chip PCR can be used to detect and precisely identify DNA of bacterial origin.
Mitterer
Georg
G
VBC-GENOMICS Bioscience Research GmbH, Vienna, Austria.
Schmidt
Wolfgang M
WM
D000818:Animals; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis*; D004275:DNA, Ribosomal / Q000032:analysis; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D019779:Salmonella enterica / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D013045:Species Specificity
D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012338:RNA, Ribosomal, 23S