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

Bradley L. Postier, Hong Liang Wang, Abhay Singh, Lori Impson, Heather L. Andrews, Jessica Klahn, Hong Li, George Risinger, David Pesta, Michael Deyholos, David W Galbraith, Louis A. Sherman, Robert L. Burnap

Research output: Contribution to journalArticle

29 Citations (Scopus)

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 PCRgenerated 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.

Original languageEnglish (US)
Article number23
JournalBMC Genomics
Volume4
DOIs
StatePublished - Jun 12 2003

Fingerprint

Bacterial DNA
Oligonucleotide Array Sequence Analysis
Polymerase Chain Reaction
DNA
Genes
Gene Expression Profiling
Genome
Gene Components
Synechocystis
Complement C3
Photosynthesis
Signal-To-Noise Ratio
Gene Library
Open Reading Frames
Organism Cloning
Proteins
Salts
Gene Expression
Costs and Cost Analysis
Temperature

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Postier, B. L., Wang, H. L., Singh, A., Impson, L., Andrews, H. L., Klahn, J., ... Burnap, R. L. (2003). The construction and use of bacterial DNA microarrays based on an optimized two-stage PCR strategy. BMC Genomics, 4, [23]. https://doi.org/10.1186/1471-2164-4-23

The construction and use of bacterial DNA microarrays based on an optimized two-stage PCR strategy. / Postier, Bradley L.; Wang, Hong Liang; Singh, Abhay; Impson, Lori; Andrews, Heather L.; Klahn, Jessica; Li, Hong; Risinger, George; Pesta, David; Deyholos, Michael; Galbraith, David W; Sherman, Louis A.; Burnap, Robert L.

In: BMC Genomics, Vol. 4, 23, 12.06.2003.

Research output: Contribution to journalArticle

Postier, BL, Wang, HL, Singh, A, Impson, L, Andrews, HL, Klahn, J, Li, H, Risinger, G, Pesta, D, Deyholos, M, Galbraith, DW, Sherman, LA & Burnap, RL 2003, 'The construction and use of bacterial DNA microarrays based on an optimized two-stage PCR strategy', BMC Genomics, vol. 4, 23. https://doi.org/10.1186/1471-2164-4-23
Postier, Bradley L. ; Wang, Hong Liang ; Singh, Abhay ; Impson, Lori ; Andrews, Heather L. ; Klahn, Jessica ; Li, Hong ; Risinger, George ; Pesta, David ; Deyholos, Michael ; Galbraith, David W ; Sherman, Louis A. ; Burnap, Robert L. / The construction and use of bacterial DNA microarrays based on an optimized two-stage PCR strategy. In: BMC Genomics. 2003 ; Vol. 4.
@article{d6b2acf6d64c48d6a8a2454ba3310657,
title = "The construction and use of bacterial DNA microarrays based on an optimized two-stage PCR strategy",
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 PCRgenerated 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.",
author = "Postier, {Bradley L.} and Wang, {Hong Liang} and Abhay Singh and Lori Impson and Andrews, {Heather L.} and Jessica Klahn and Hong Li and George Risinger and David Pesta and Michael Deyholos and Galbraith, {David W} and Sherman, {Louis A.} and Burnap, {Robert L.}",
year = "2003",
month = "6",
day = "12",
doi = "10.1186/1471-2164-4-23",
language = "English (US)",
volume = "4",
journal = "BMC Genomics",
issn = "1471-2164",
publisher = "BioMed Central",

}

TY - JOUR

T1 - The construction and use of bacterial DNA microarrays based on an optimized two-stage PCR strategy

AU - Postier, Bradley L.

AU - Wang, Hong Liang

AU - Singh, Abhay

AU - Impson, Lori

AU - Andrews, Heather L.

AU - Klahn, Jessica

AU - Li, Hong

AU - Risinger, George

AU - Pesta, David

AU - Deyholos, Michael

AU - Galbraith, David W

AU - Sherman, Louis A.

AU - Burnap, Robert L.

PY - 2003/6/12

Y1 - 2003/6/12

N2 - 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 PCRgenerated 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.

AB - 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 PCRgenerated 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.

UR - http://www.scopus.com/inward/record.url?scp=9444248636&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=9444248636&partnerID=8YFLogxK

U2 - 10.1186/1471-2164-4-23

DO - 10.1186/1471-2164-4-23

M3 - Article

C2 - 12803655

AN - SCOPUS:9444248636

VL - 4

JO - BMC Genomics

JF - BMC Genomics

SN - 1471-2164

M1 - 23

ER -