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An Integrated PCR Colony Hybridization Approach to Screen cDNA Libraries for Full-Length Coding Sequences

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 expr...

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Autores principales: Pollier, Jacob, González-Guzmán, Miguel, Ardiles-Diaz, Wilson, Geelen, Danny, Goossens, Alain
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3174253/
https://www.ncbi.nlm.nih.gov/pubmed/21949817
http://dx.doi.org/10.1371/journal.pone.0024978
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author Pollier, Jacob
González-Guzmán, Miguel
Ardiles-Diaz, Wilson
Geelen, Danny
Goossens, Alain
author_facet Pollier, Jacob
González-Guzmán, Miguel
Ardiles-Diaz, Wilson
Geelen, Danny
Goossens, Alain
author_sort Pollier, Jacob
collection PubMed
description 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.
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spelling pubmed-31742532011-09-26 An Integrated PCR Colony Hybridization Approach to Screen cDNA Libraries for Full-Length Coding Sequences Pollier, Jacob González-Guzmán, Miguel Ardiles-Diaz, Wilson Geelen, Danny Goossens, Alain PLoS One Research Article 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. Public Library of Science 2011-09-15 /pmc/articles/PMC3174253/ /pubmed/21949817 http://dx.doi.org/10.1371/journal.pone.0024978 Text en Pollier et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Pollier, Jacob
González-Guzmán, Miguel
Ardiles-Diaz, Wilson
Geelen, Danny
Goossens, Alain
An Integrated PCR Colony Hybridization Approach to Screen cDNA Libraries for Full-Length Coding Sequences
title An Integrated PCR Colony Hybridization Approach to Screen cDNA Libraries for Full-Length Coding Sequences
title_full An Integrated PCR Colony Hybridization Approach to Screen cDNA Libraries for Full-Length Coding Sequences
title_fullStr An Integrated PCR Colony Hybridization Approach to Screen cDNA Libraries for Full-Length Coding Sequences
title_full_unstemmed An Integrated PCR Colony Hybridization Approach to Screen cDNA Libraries for Full-Length Coding Sequences
title_short An Integrated PCR Colony Hybridization Approach to Screen cDNA Libraries for Full-Length Coding Sequences
title_sort integrated pcr colony hybridization approach to screen cdna libraries for full-length coding sequences
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3174253/
https://www.ncbi.nlm.nih.gov/pubmed/21949817
http://dx.doi.org/10.1371/journal.pone.0024978
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