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Negative Subtraction Hybridization: An efficient method to isolate large numbers of condition-specific cDNAs

BACKGROUND: The construction of cDNA libraries is a useful tool to understand gene expression in organisms under different conditions, but random sequencing of unbiased cDNA collections is laborious and can give rise to redundant EST collections. We aimed to isolate cDNAs of messages induced by swit...

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Autores principales: Ray, Anamika, Macwana, Sunita, Ayoubi, Patricia, Hall, Leo T, Prade, Rolf, Mort, Andrew J
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2004
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC400731/
https://www.ncbi.nlm.nih.gov/pubmed/15050035
http://dx.doi.org/10.1186/1471-2164-5-22
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author Ray, Anamika
Macwana, Sunita
Ayoubi, Patricia
Hall, Leo T
Prade, Rolf
Mort, Andrew J
author_facet Ray, Anamika
Macwana, Sunita
Ayoubi, Patricia
Hall, Leo T
Prade, Rolf
Mort, Andrew J
author_sort Ray, Anamika
collection PubMed
description BACKGROUND: The construction of cDNA libraries is a useful tool to understand gene expression in organisms under different conditions, but random sequencing of unbiased cDNA collections is laborious and can give rise to redundant EST collections. We aimed to isolate cDNAs of messages induced by switching Aspergillus nidulans from growth on glucose to growth on selected polysaccharides. Approximately 4,700 contigs from 12,320 ESTs were already available from a cDNA library representing transcripts isolated from glucose-grown A. nidulans during asexual development. Our goals were to expand the cDNA collection without repeated sequencing of previously identified ESTs and to find as many transcripts as possible that are specifically induced in complex polysaccharide metabolism. RESULTS: We have devised a Negative Subtraction Hybridization (NSH) method and tested it in A. nidulans. NSH entails screening a plasmid library made from cDNAs prepared from cells grown under a selected physiological condition with labeled cDNA probes prepared from another physiological condition. Plasmids with inserts that failed to hybridize to cDNA probes through two rounds of screening (i.e. negatives) indicate that they are transcripts present at low concentration in the labeled probe pool. Thus, these transcripts will be predominantly condition-specific, along with some rare transcripts. In a screen for transcripts induced by switching the carbon source from glucose to 12 selected polysaccharides, 3,532 negatives were isolated from approximately 100,000 surveyed colonies using this method. Negative clones were end-sequenced and assembled into 2,039 contigs, of which 1,722 were not present in the previously characterized glucose-grown cDNA library. Single-channel microarray hybridization experiments confirmed that the majority of the negatives represented genes that were differentially induced by a switch from growth in glucose to one or more of the polysaccharides. CONCLUSIONS: The Negative Subtraction Hybridization method described here has several practical benefits. This method can be used to screen any existing cDNA library, including full-length and pooled libraries, and does not rely on PCR or sequence information. In addition, NSH is a cost-effective method for the isolation of novel, full-length cDNAs for differentially expressed transcripts or enrichment of rare transcripts.
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spelling pubmed-4007312004-05-02 Negative Subtraction Hybridization: An efficient method to isolate large numbers of condition-specific cDNAs Ray, Anamika Macwana, Sunita Ayoubi, Patricia Hall, Leo T Prade, Rolf Mort, Andrew J BMC Genomics Methodology Article BACKGROUND: The construction of cDNA libraries is a useful tool to understand gene expression in organisms under different conditions, but random sequencing of unbiased cDNA collections is laborious and can give rise to redundant EST collections. We aimed to isolate cDNAs of messages induced by switching Aspergillus nidulans from growth on glucose to growth on selected polysaccharides. Approximately 4,700 contigs from 12,320 ESTs were already available from a cDNA library representing transcripts isolated from glucose-grown A. nidulans during asexual development. Our goals were to expand the cDNA collection without repeated sequencing of previously identified ESTs and to find as many transcripts as possible that are specifically induced in complex polysaccharide metabolism. RESULTS: We have devised a Negative Subtraction Hybridization (NSH) method and tested it in A. nidulans. NSH entails screening a plasmid library made from cDNAs prepared from cells grown under a selected physiological condition with labeled cDNA probes prepared from another physiological condition. Plasmids with inserts that failed to hybridize to cDNA probes through two rounds of screening (i.e. negatives) indicate that they are transcripts present at low concentration in the labeled probe pool. Thus, these transcripts will be predominantly condition-specific, along with some rare transcripts. In a screen for transcripts induced by switching the carbon source from glucose to 12 selected polysaccharides, 3,532 negatives were isolated from approximately 100,000 surveyed colonies using this method. Negative clones were end-sequenced and assembled into 2,039 contigs, of which 1,722 were not present in the previously characterized glucose-grown cDNA library. Single-channel microarray hybridization experiments confirmed that the majority of the negatives represented genes that were differentially induced by a switch from growth in glucose to one or more of the polysaccharides. CONCLUSIONS: The Negative Subtraction Hybridization method described here has several practical benefits. This method can be used to screen any existing cDNA library, including full-length and pooled libraries, and does not rely on PCR or sequence information. In addition, NSH is a cost-effective method for the isolation of novel, full-length cDNAs for differentially expressed transcripts or enrichment of rare transcripts. BioMed Central 2004-03-29 /pmc/articles/PMC400731/ /pubmed/15050035 http://dx.doi.org/10.1186/1471-2164-5-22 Text en Copyright © 2004 Ray 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.
spellingShingle Methodology Article
Ray, Anamika
Macwana, Sunita
Ayoubi, Patricia
Hall, Leo T
Prade, Rolf
Mort, Andrew J
Negative Subtraction Hybridization: An efficient method to isolate large numbers of condition-specific cDNAs
title Negative Subtraction Hybridization: An efficient method to isolate large numbers of condition-specific cDNAs
title_full Negative Subtraction Hybridization: An efficient method to isolate large numbers of condition-specific cDNAs
title_fullStr Negative Subtraction Hybridization: An efficient method to isolate large numbers of condition-specific cDNAs
title_full_unstemmed Negative Subtraction Hybridization: An efficient method to isolate large numbers of condition-specific cDNAs
title_short Negative Subtraction Hybridization: An efficient method to isolate large numbers of condition-specific cDNAs
title_sort negative subtraction hybridization: an efficient method to isolate large numbers of condition-specific cdnas
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC400731/
https://www.ncbi.nlm.nih.gov/pubmed/15050035
http://dx.doi.org/10.1186/1471-2164-5-22
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