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...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2011-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3174253?pdf=render |
| _version_ | 1818138118993215488 |
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| author | Jacob Pollier Miguel González-Guzmán Wilson Ardiles-Diaz Danny Geelen Alain Goossens |
| author_facet | Jacob Pollier Miguel González-Guzmán Wilson Ardiles-Diaz Danny Geelen Alain Goossens |
| author_sort | Jacob Pollier |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-11T10:07:07Z |
| format | Article |
| id | doaj.art-b0405424b2fd44a6bf99807d513fd25d |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-11T10:07:07Z |
| publishDate | 2011-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-b0405424b2fd44a6bf99807d513fd25d2022-12-22T01:11:54ZengPublic Library of Science (PLoS)PLoS ONE1932-62032011-01-0169e2497810.1371/journal.pone.0024978An integrated PCR colony hybridization approach to screen cDNA libraries for full-length coding sequences.Jacob PollierMiguel González-GuzmánWilson Ardiles-DiazDanny GeelenAlain GoossensBACKGROUND: 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.http://europepmc.org/articles/PMC3174253?pdf=render |
| spellingShingle | Jacob Pollier Miguel González-Guzmán Wilson Ardiles-Diaz Danny Geelen Alain Goossens An integrated PCR colony hybridization approach to screen cDNA libraries for full-length coding sequences. PLoS ONE |
| 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 |
| url | http://europepmc.org/articles/PMC3174253?pdf=render |
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