The construction and use of bacterial DNA microarrays based on an optimized two-stage PCR strategy

Persistent Link:
http://hdl.handle.net/10150/610394
Title:
The construction and use of bacterial DNA microarrays based on an optimized two-stage PCR strategy
Author:
Postier, Bradley; Wang, Hong-Liang; Singh, Abhay; Impson, Lori; Andrews, Heather; Klahn, Jessica; Li, Hong; Risinger, George; Pesta, David; Deyholos, Michael; Galbraith, David; Sherman, Louis; Burnap, Robert
Affiliation:
Department of Microbiology & Molecular Genetics, Oklahoma State University, Stillwater, OK 74078 USA; Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA; Department of Plant Sciences, University of Arizona, Tucson AZ 85721 USA
Issue Date:
2003
Publisher:
BioMed Central
Citation:
BMC Genomics 2003, 4:23 http://www.biomedcentral.com/1471-2164/4/23
Journal:
BMC Genomics
Rights:
© 2003 Postier et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.
Collection Information:
This item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at repository@u.library.arizona.edu.
Abstract:
BACKGROUND:DNA microarrays are a powerful tool with important applications such as global gene expression profiling. Construction of bacterial DNA microarrays from genomic sequence data using a two-stage PCR amplification approach for the production of arrayed DNA is attractive because it allows, in principal, the continued re-amplification of DNA fragments and facilitates further utilization of the DNA fragments for additional uses (e.g. over-expression of protein). We describe the successful construction and use of DNA microarrays by the two-stage amplification approach and discuss the technical challenges that were met and resolved during the project.RESULTS:Chimeric primers that contained both gene-specific and shared, universal sequence allowed the two-stage amplification of the 3,168 genes identified on the genome of Synechocystis sp. PCC6803, an important prokaryotic model organism for the study of oxygenic photosynthesis. The gene-specific component of the primer was of variable length to maintain uniform annealing temperatures during the 1st round of PCR synthesis, and situated to preserve full-length ORFs. Genes were truncated at 2 kb for efficient amplification, so that about 92% of the PCR fragments were full-length genes. The two-stage amplification had the additional advantage of normalizing the yield of PCR products and this improved the uniformity of DNA features robotically deposited onto the microarray surface. We also describe the techniques utilized to optimize hybridization conditions and signal-to-noise ratio of the transcription profile. The inter-lab transportability was demonstrated by the virtual error-free amplification of the entire genome complement of 3,168 genes using the universal primers in partner labs. The printed slides have been successfully used to identify differentially expressed genes in response to a number of environmental conditions, including salt stress.CONCLUSIONS:The technique detailed here minimizes the cost and effort to replicate a PCR-generated DNA gene fragment library and facilitates several downstream processes (e.g. directional cloning of fragments and gene expression as affinity-tagged fusion proteins) beyond the primary objective of producing DNA microarrays for global gene expression profiling.
EISSN:
1471-2164
DOI:
10.1186/1471-2164-4-23
Version:
Final published version
Additional Links:
http://www.biomedcentral.com/1471-2164/4/23

Full metadata record

DC FieldValue Language
dc.contributor.authorPostier, Bradleyen
dc.contributor.authorWang, Hong-Liangen
dc.contributor.authorSingh, Abhayen
dc.contributor.authorImpson, Lorien
dc.contributor.authorAndrews, Heatheren
dc.contributor.authorKlahn, Jessicaen
dc.contributor.authorLi, Hongen
dc.contributor.authorRisinger, Georgeen
dc.contributor.authorPesta, Daviden
dc.contributor.authorDeyholos, Michaelen
dc.contributor.authorGalbraith, Daviden
dc.contributor.authorSherman, Louisen
dc.contributor.authorBurnap, Roberten
dc.date.accessioned2016-05-20T09:05:50Z-
dc.date.available2016-05-20T09:05:50Z-
dc.date.issued2003en
dc.identifier.citationBMC Genomics 2003, 4:23 http://www.biomedcentral.com/1471-2164/4/23en
dc.identifier.doi10.1186/1471-2164-4-23en
dc.identifier.urihttp://hdl.handle.net/10150/610394-
dc.description.abstractBACKGROUND:DNA microarrays are a powerful tool with important applications such as global gene expression profiling. Construction of bacterial DNA microarrays from genomic sequence data using a two-stage PCR amplification approach for the production of arrayed DNA is attractive because it allows, in principal, the continued re-amplification of DNA fragments and facilitates further utilization of the DNA fragments for additional uses (e.g. over-expression of protein). We describe the successful construction and use of DNA microarrays by the two-stage amplification approach and discuss the technical challenges that were met and resolved during the project.RESULTS:Chimeric primers that contained both gene-specific and shared, universal sequence allowed the two-stage amplification of the 3,168 genes identified on the genome of Synechocystis sp. PCC6803, an important prokaryotic model organism for the study of oxygenic photosynthesis. The gene-specific component of the primer was of variable length to maintain uniform annealing temperatures during the 1st round of PCR synthesis, and situated to preserve full-length ORFs. Genes were truncated at 2 kb for efficient amplification, so that about 92% of the PCR fragments were full-length genes. The two-stage amplification had the additional advantage of normalizing the yield of PCR products and this improved the uniformity of DNA features robotically deposited onto the microarray surface. We also describe the techniques utilized to optimize hybridization conditions and signal-to-noise ratio of the transcription profile. The inter-lab transportability was demonstrated by the virtual error-free amplification of the entire genome complement of 3,168 genes using the universal primers in partner labs. The printed slides have been successfully used to identify differentially expressed genes in response to a number of environmental conditions, including salt stress.CONCLUSIONS:The technique detailed here minimizes the cost and effort to replicate a PCR-generated DNA gene fragment library and facilitates several downstream processes (e.g. directional cloning of fragments and gene expression as affinity-tagged fusion proteins) beyond the primary objective of producing DNA microarrays for global gene expression profiling.en
dc.language.isoenen
dc.publisherBioMed Centralen
dc.relation.urlhttp://www.biomedcentral.com/1471-2164/4/23en
dc.rights© 2003 Postier et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.en
dc.titleThe construction and use of bacterial DNA microarrays based on an optimized two-stage PCR strategyen
dc.typeArticleen
dc.identifier.eissn1471-2164en
dc.contributor.departmentDepartment of Microbiology & Molecular Genetics, Oklahoma State University, Stillwater, OK 74078 USAen
dc.contributor.departmentDepartment of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USAen
dc.contributor.departmentDepartment of Plant Sciences, University of Arizona, Tucson AZ 85721 USAen
dc.identifier.journalBMC Genomicsen
dc.description.collectioninformationThis item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at repository@u.library.arizona.edu.en
dc.eprint.versionFinal published versionen
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