A simple and efficient method for purification of multiple cDNAs by using a single hybridization step on nylon discs

A simple and rapid technique for purification of multiple cDNAs and for subsequent cloning was developed. The procedure involves first-strand cDNA synthesis using oligo-dT as a primer, followed by removal of the mRNA by alkali hydrolysis and tailing of the cDNA at the 3' end with dC residues (a...

Full description

Saved in:
Bibliographic Details
Published in:Plant molecular biology 1992-10, Vol.20 (1), p.31-35
Main Authors: Pramanik, S.K. (Guelph Univ., Guelph, Ont. (Canada). Dept. of Botany), Bewley, J.D
Format: Article
Language:English
Subjects:
ADN
Analytical, structural and metabolic biochemistry
Base Sequence
Biological and medical sciences
CLONACION MOLECULAR
CLONAGE MOLECULAIRE
Cloning, Molecular - methods
DNA
DNA - genetics
DNA - isolation & purification
DNA, Single-Stranded - genetics
DNA, Single-Stranded - isolation & purification
Fundamental and applied biological sciences. Psychology
General aspects, investigation methods
HIBRIDACION MOLECULAR
HYBRIDATION MOLECULAIRE
METHODE
METHODS
METODOS
MOLECULAR CLONING
MOLECULAR HYBRIDIZATION
Molecular Sequence Data
Molecular Weight
Nucleic Acid Hybridization
Nucleic acids
Nylons
Oligodeoxyribonucleotides
Plants - genetics
Polymerase Chain Reaction - methods
PURIFICACION
PURIFICATION
Citations: Items that this one cites
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:A simple and rapid technique for purification of multiple cDNAs and for subsequent cloning was developed. The procedure involves first-strand cDNA synthesis using oligo-dT as a primer, followed by removal of the mRNA by alkali hydrolysis and tailing of the cDNA at the 3' end with dC residues (about 18-20 residues). Single-stranded cDNA species can be isolated from this dC-tailed single-stranded cDNA population (dC-ss-cDNA) by hybrid selection. The specific probes are immobilized on individual nylon discs and hybridized in bulk against the dC-ss-cDNA population. After washing under appropriate stringency conditions, the hybrid-selected dC-ss-cDNAs are eluted separately from each nylon disc. These cDNAs are then available for amplification by PCR (if a rare species) using oligo-dG and oligo-dT as upstream and downstream primers, respectively, or in the case of an abundant message, the cDNAs can be used directly for second-strand synthesis using oligo-dG as the primer. The advantage of this technique is that a large number of different cDNAs can be screened easily and economically in a single hybridization reaction.
ISSN:0167-4412
1573-5028
DOI:10.1007/BF00029146