Chemiluminescent detection of sequential DNA hybridizations to high- density, filter-arrayed cDNA libraries: A subtraction method for novel gene discovery

D. Guiliano, M. Ganatra, J. Ware, J. Parrot, J. Daub, L. Moran, H. Brennecke, J. M. Foster, T. Supali, M. Blaxter, Alan Leroy Scott, S. A. Williams, B. E. Slatko

Research output: Contribution to journalArticle

Abstract

A chemiluminescent approach for sequential DNA hybridizations to high- density filter arrays of cDNAs, using a biotin-based random priming method followed by a streptavidin/alkaline phosphatase/CDP-Star(TM) detection protocol, is presented. The method has been applied to the Brugia malayi genome project, wherein cDNA libraries, cosmid and bacterial artificial chromosome (BAC) libraries have been gridded at high density onto nylon filters for subsequent analysis by hybridization. Individual probes and pools of rRNA probes, ribosomal protein probes and expressed sequence tag probes show correct specificity and high signal-to-noise ratios even after ten rounds of hybridization, detection, stripping of the probes from the membranes and rehybridization with additional probe sets. This approach provides a subtraction method that leads to a reduction in redundant DNA sequencing, thus increasing the rate of novel gene discovery. The method is also applicable for detecting target sequences, which are present in one or only a few copies per cell; it has proven useful for physical mapping of BAC and cosmid high-density filter arrays, wherein multiple probes have been hybridized at one time (multiplexed) and subsequently 'deplexed' into individual components for specific probe localizations.

Original languageEnglish (US)
Pages (from-to)146-152
Number of pages7
JournalBioTechniques
Volume27
Issue number1
StatePublished - 1999

Fingerprint

Genetic Association Studies
Gene Library
Genes
Bacterial Artificial Chromosomes
Cosmids
DNA
Brugia malayi
Cytidine Diphosphate
Streptavidin
Ribosomal Proteins
Nylons
Expressed Sequence Tags
Signal-To-Noise Ratio
Chromosomes
Biotin
Oligonucleotide Array Sequence Analysis
DNA Sequence Analysis
Alkaline Phosphatase
Genome
Membranes

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Guiliano, D., Ganatra, M., Ware, J., Parrot, J., Daub, J., Moran, L., ... Slatko, B. E. (1999). Chemiluminescent detection of sequential DNA hybridizations to high- density, filter-arrayed cDNA libraries: A subtraction method for novel gene discovery. BioTechniques, 27(1), 146-152.

Chemiluminescent detection of sequential DNA hybridizations to high- density, filter-arrayed cDNA libraries : A subtraction method for novel gene discovery. / Guiliano, D.; Ganatra, M.; Ware, J.; Parrot, J.; Daub, J.; Moran, L.; Brennecke, H.; Foster, J. M.; Supali, T.; Blaxter, M.; Scott, Alan Leroy; Williams, S. A.; Slatko, B. E.

In: BioTechniques, Vol. 27, No. 1, 1999, p. 146-152.

Research output: Contribution to journalArticle

Guiliano, D, Ganatra, M, Ware, J, Parrot, J, Daub, J, Moran, L, Brennecke, H, Foster, JM, Supali, T, Blaxter, M, Scott, AL, Williams, SA & Slatko, BE 1999, 'Chemiluminescent detection of sequential DNA hybridizations to high- density, filter-arrayed cDNA libraries: A subtraction method for novel gene discovery', BioTechniques, vol. 27, no. 1, pp. 146-152.
Guiliano, D. ; Ganatra, M. ; Ware, J. ; Parrot, J. ; Daub, J. ; Moran, L. ; Brennecke, H. ; Foster, J. M. ; Supali, T. ; Blaxter, M. ; Scott, Alan Leroy ; Williams, S. A. ; Slatko, B. E. / Chemiluminescent detection of sequential DNA hybridizations to high- density, filter-arrayed cDNA libraries : A subtraction method for novel gene discovery. In: BioTechniques. 1999 ; Vol. 27, No. 1. pp. 146-152.
@article{d1c09af1dfcd4547840391a6f3324efe,
title = "Chemiluminescent detection of sequential DNA hybridizations to high- density, filter-arrayed cDNA libraries: A subtraction method for novel gene discovery",
abstract = "A chemiluminescent approach for sequential DNA hybridizations to high- density filter arrays of cDNAs, using a biotin-based random priming method followed by a streptavidin/alkaline phosphatase/CDP-Star(TM) detection protocol, is presented. The method has been applied to the Brugia malayi genome project, wherein cDNA libraries, cosmid and bacterial artificial chromosome (BAC) libraries have been gridded at high density onto nylon filters for subsequent analysis by hybridization. Individual probes and pools of rRNA probes, ribosomal protein probes and expressed sequence tag probes show correct specificity and high signal-to-noise ratios even after ten rounds of hybridization, detection, stripping of the probes from the membranes and rehybridization with additional probe sets. This approach provides a subtraction method that leads to a reduction in redundant DNA sequencing, thus increasing the rate of novel gene discovery. The method is also applicable for detecting target sequences, which are present in one or only a few copies per cell; it has proven useful for physical mapping of BAC and cosmid high-density filter arrays, wherein multiple probes have been hybridized at one time (multiplexed) and subsequently 'deplexed' into individual components for specific probe localizations.",
author = "D. Guiliano and M. Ganatra and J. Ware and J. Parrot and J. Daub and L. Moran and H. Brennecke and Foster, {J. M.} and T. Supali and M. Blaxter and Scott, {Alan Leroy} and Williams, {S. A.} and Slatko, {B. E.}",
year = "1999",
language = "English (US)",
volume = "27",
pages = "146--152",
journal = "BioTechniques",
issn = "0736-6205",
publisher = "Eaton Publishing Company",
number = "1",

}

TY - JOUR

T1 - Chemiluminescent detection of sequential DNA hybridizations to high- density, filter-arrayed cDNA libraries

T2 - A subtraction method for novel gene discovery

AU - Guiliano, D.

AU - Ganatra, M.

AU - Ware, J.

AU - Parrot, J.

AU - Daub, J.

AU - Moran, L.

AU - Brennecke, H.

AU - Foster, J. M.

AU - Supali, T.

AU - Blaxter, M.

AU - Scott, Alan Leroy

AU - Williams, S. A.

AU - Slatko, B. E.

PY - 1999

Y1 - 1999

N2 - A chemiluminescent approach for sequential DNA hybridizations to high- density filter arrays of cDNAs, using a biotin-based random priming method followed by a streptavidin/alkaline phosphatase/CDP-Star(TM) detection protocol, is presented. The method has been applied to the Brugia malayi genome project, wherein cDNA libraries, cosmid and bacterial artificial chromosome (BAC) libraries have been gridded at high density onto nylon filters for subsequent analysis by hybridization. Individual probes and pools of rRNA probes, ribosomal protein probes and expressed sequence tag probes show correct specificity and high signal-to-noise ratios even after ten rounds of hybridization, detection, stripping of the probes from the membranes and rehybridization with additional probe sets. This approach provides a subtraction method that leads to a reduction in redundant DNA sequencing, thus increasing the rate of novel gene discovery. The method is also applicable for detecting target sequences, which are present in one or only a few copies per cell; it has proven useful for physical mapping of BAC and cosmid high-density filter arrays, wherein multiple probes have been hybridized at one time (multiplexed) and subsequently 'deplexed' into individual components for specific probe localizations.

AB - A chemiluminescent approach for sequential DNA hybridizations to high- density filter arrays of cDNAs, using a biotin-based random priming method followed by a streptavidin/alkaline phosphatase/CDP-Star(TM) detection protocol, is presented. The method has been applied to the Brugia malayi genome project, wherein cDNA libraries, cosmid and bacterial artificial chromosome (BAC) libraries have been gridded at high density onto nylon filters for subsequent analysis by hybridization. Individual probes and pools of rRNA probes, ribosomal protein probes and expressed sequence tag probes show correct specificity and high signal-to-noise ratios even after ten rounds of hybridization, detection, stripping of the probes from the membranes and rehybridization with additional probe sets. This approach provides a subtraction method that leads to a reduction in redundant DNA sequencing, thus increasing the rate of novel gene discovery. The method is also applicable for detecting target sequences, which are present in one or only a few copies per cell; it has proven useful for physical mapping of BAC and cosmid high-density filter arrays, wherein multiple probes have been hybridized at one time (multiplexed) and subsequently 'deplexed' into individual components for specific probe localizations.

UR - http://www.scopus.com/inward/record.url?scp=0032987748&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032987748&partnerID=8YFLogxK

M3 - Article

C2 - 10407677

AN - SCOPUS:0032987748

VL - 27

SP - 146

EP - 152

JO - BioTechniques

JF - BioTechniques

SN - 0736-6205

IS - 1

ER -