An integrated PCR colony hybridization approach to screen cDNA libraries for full-length coding sequences
- Author
- Jacob Pollier (UGent) , Miguel Gonzalez Guzman (UGent) , Wilson Ardiles Diaz (UGent) , Danny Geelen (UGent) and Alain Goossens (UGent)
- Organization
- Abstract
- Background: cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) is a commonly used technique for genome-wide expression analysis that does not require prior sequence knowledge. Typically, quantitative expression data and sequence information are obtained for a large number of differentially expressed gene tags. However, most of the gene tags do not correspond to full-length (FL) coding sequences, which is a prerequisite for subsequent functional analysis. Methodology: A medium-throughput screening strategy, based on integration of polymerase chain reaction (PCR) and colony hybridization, was developed that allows in parallel screening of a cDNA library for FL clones corresponding to incomplete cDNAs. The method was applied to screen for the FL open reading frames of a selection of 163 cDNA-AFLP tags from three different medicinal plants, leading to the identification of 109 (67%) FL clones. Furthermore, the protocol allows for the use of multiple probes in a single hybridization event, thus significantly increasing the throughput when screening for rare transcripts. Conclusions: The presented strategy offers an efficient method for the conversion of incomplete expressed sequence tags (ESTs), such as cDNA-AFLP tags, to FL-coding sequences.
- Keywords
- GENOME-WIDE EXPRESSION, FAMILY, AMPLIFICATION, AFLP
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Citation
Please use this url to cite or link to this publication: http://hdl.handle.net/1854/LU-1939649
- Chicago
- Pollier, Jacob, Miguel Gonzalez Guzman, Wilson Ardiles Diaz, Danny Geelen, and Alain Goossens. 2011. “An Integrated PCR Colony Hybridization Approach to Screen cDNA Libraries for Full-length Coding Sequences.” Plos One 6 (9).
- APA
- Pollier, J., Gonzalez Guzman, M., Ardiles Diaz, W., Geelen, D., & Goossens, A. (2011). An integrated PCR colony hybridization approach to screen cDNA libraries for full-length coding sequences. PLOS ONE, 6(9).
- Vancouver
- 1.Pollier J, Gonzalez Guzman M, Ardiles Diaz W, Geelen D, Goossens A. An integrated PCR colony hybridization approach to screen cDNA libraries for full-length coding sequences. PLOS ONE. 2011;6(9).
- MLA
- Pollier, Jacob, Miguel Gonzalez Guzman, Wilson Ardiles Diaz, et al. “An Integrated PCR Colony Hybridization Approach to Screen cDNA Libraries for Full-length Coding Sequences.” PLOS ONE 6.9 (2011): n. pag. Print.
@article{1939649,
abstract = {Background: cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) is a commonly used technique for genome-wide expression analysis that does not require prior sequence knowledge. Typically, quantitative expression data and sequence information are obtained for a large number of differentially expressed gene tags. However, most of the gene tags do not correspond to full-length (FL) coding sequences, which is a prerequisite for subsequent functional analysis.
Methodology: A medium-throughput screening strategy, based on integration of polymerase chain reaction (PCR) and colony hybridization, was developed that allows in parallel screening of a cDNA library for FL clones corresponding to incomplete cDNAs. The method was applied to screen for the FL open reading frames of a selection of 163 cDNA-AFLP tags from three different medicinal plants, leading to the identification of 109 (67\%) FL clones. Furthermore, the protocol allows for the use of multiple probes in a single hybridization event, thus significantly increasing the throughput when screening for rare transcripts.
Conclusions: The presented strategy offers an efficient method for the conversion of incomplete expressed sequence tags (ESTs), such as cDNA-AFLP tags, to FL-coding sequences.},
articleno = {e24978},
author = {Pollier, Jacob and Gonzalez Guzman, Miguel and Ardiles Diaz, Wilson and Geelen, Danny and Goossens, Alain},
issn = {1932-6203},
journal = {PLOS ONE},
language = {eng},
number = {9},
pages = {6},
title = {An integrated PCR colony hybridization approach to screen cDNA libraries for full-length coding sequences},
url = {http://dx.doi.org/10.1371/journal.pone.0024978},
volume = {6},
year = {2011},
}
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