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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. 25 figs.

Inventors:
;
Issue Date:
Research Org.:
Columbia Univ., New York, NY (United States)
Sponsoring Org.:
USDOE, Washington, DC (United States)
OSTI Identifier:
321194
Patent Number(s):
5846721
Application Number:
PAN: 8-715,941; CNN: Grant 1RO1HG00980
Assignee:
Columbia Univ., New York, NY (United States)
DOE Contract Number:  
FG02-91ER61233
Resource Type:
Patent
Resource Relation:
Other Information: PBD: 8 Dec 1998
Country of Publication:
United States
Language:
English
Subject:
55 BIOLOGY AND MEDICINE, BASIC STUDIES; DNA-CLONING; MOLECULAR BIOLOGY; POLYMERASE CHAIN REACTION; DNA

Citation Formats

Soares, M B, and Fatima Bonaldo, M de. Efficient and simpler method to construct normalized cDNA libraries with improved representations of full-length cDNAs. United States: N. p., 1998. Web.
Soares, M B, & Fatima Bonaldo, M de. Efficient and simpler method to construct normalized cDNA libraries with improved representations of full-length cDNAs. United States.
Soares, M B, and Fatima Bonaldo, M de. Tue . "Efficient and simpler method to construct normalized cDNA libraries with improved representations of full-length cDNAs". United States.
@article{osti_321194,
title = {Efficient and simpler method to construct normalized cDNA libraries with improved representations of full-length cDNAs},
author = {Soares, M B and Fatima Bonaldo, M de},
abstractNote = {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. 25 figs.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {1998},
month = {12}
}