Title: Efficient and simpler method to construct normalized cDNA libraries with improved representations of full-length cDNAs

Abstract

This invention provides a method to normalize a cDNA library comprising: (a) constructing a directionally cloned library containing cDNA inserts wherein the insert is capable of being amplified by polymerase chain reaction; (b) converting a double-stranded cDNA library into single-stranded DNA circles; (c) generating single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) by polymerase chain reaction with appropriate primers; (d) hybridizing the single-stranded DNA circles converted in step (b) with the complementary single-stranded nucleic acid molecules generated in step (c) to produce partial duplexes to an appropriate Cot; and (e) separating the unhybridized single-stranded DNA circles from the hybridized DNA circles, thereby generating a normalized cDNA library. This invention also provides a method to normalize a cDNA library wherein the generating of single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) is by excising cDNA inserts from the double-stranded cDNA library; purifying the cDNA inserts from cloning vectors; and digesting the cDNA inserts with an exonuclease. This invention further provides a method to construct a subtractive cDNA library following the steps described above. This invention further provides normalized and/or subtractive cDNA libraries generated by the above methods.

Inventors:

Soares, Marcelo Bento ^[1]; Bonaldo, Maria de Fatima ^[1]

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1. New York, NY

Issue Date:

Thu Jan 01 00:00:00 EST 1998

Research Org.:

Columbia Univ., New York, NY (United States)

OSTI Identifier:

872025

Patent Number(s):

5846721

Assignee:

Trustees of Columbia University in City of New York (New York, NY)

Patent Classifications (CPCs):

C - CHEMISTRY C12 - BIOCHEMISTRY C12N - MICROORGANISMS OR ENZYMES

C - CHEMISTRY C12 - BIOCHEMISTRY C12Q - MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS

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C - CHEMISTRY C12 - BIOCHEMISTRY C12N - MICROORGANISMS OR ENZYMES

C12N15/1072 - {Differential gene expression library synthesis, e.g. subtracted libraries, differential screening}
C12N15/1093 - {General methods of preparing gene libraries, not provided for in other subgroups}

C - CHEMISTRY C12 - BIOCHEMISTRY C12Q - MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS

C12Q1/6809 - Methods for determination or identification of nucleic acids involving differential detection

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DOE Contract Number:  

FG02-91ER61233

Resource Type:

Patent

Country of Publication:

United States

Language:

English

Subject:

efficient; simpler; method; construct; normalized; cdna; libraries; improved; representations; full-length; cdnas; provides; normalize; library; comprising; constructing; directionally; cloned; containing; inserts; insert; capable; amplified; polymerase; chain; reaction; converting; double-stranded; single-stranded; dna; circles; generating; nucleic; acid; molecules; complementary; converted; step; appropriate; primers; hybridizing; generated; produce; partial; duplexes; cot; separating; unhybridized; hybridized; excising; purifying; cloning; vectors; digesting; exonuclease; subtractive; following; steps; described; methods; acid molecule; cdna libraries; hybridized dna; stranded dna; produce partial; chain reaction; acid molecules; nucleic acid; polymerase chain; cdna library; single-stranded nucleic; cloning vector; partial duplexes; single-stranded dna; normalized cdna; appropriate cot; molecules complementary; full-length cdna; containing cdna; stranded nucleic; dna insert; /435/999/