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 = {Tue Dec 08 00:00:00 EST 1998},
month = {Tue Dec 08 00:00:00 EST 1998}
}