Microarray-based Detection of Bacteria by On-chip Pcr.
From: VBC-GENOMICS Bioscience Research GmbH, Vienna, Austria.
Methods in molecular biology (Clifton, N.J.)
- Publish Date: 2006
- ISSN: 1064-3745
- Volume: 345
- Issue:
- Pages: 37-51
- Medium: Print
- Language: English
- Citation (JAMA): Mitterer Georg, Schmidt Wolfgang M, et al. Microarray-based Detection of Bacteria by On-chip Pcr.. Methods Mol. Biol. 2006;345:37-51
Abstract
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.
Mesh Headings (Keywords): Animals, DNA Primers, DNA, Bacterial, DNA, Ribosomal, Humans, Oligonucleotide Array Sequence Analysis, Polymerase Chain Reaction, RNA, Ribosomal, 23S, Salmonella enterica, Species Specificity
Check for Full Text / PubMed Unique Identifier (PMID): 16957345
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