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

Patent ·
OSTI ID:872025
 [1];  [1]
  1. New York, NY
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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.

Research Organization:
Columbia University
DOE Contract Number:
FG02-91ER61233
Assignee:
Trustees of Columbia University in City of New York (New York, NY)
Patent Number(s):
US 5846721
OSTI ID:
872025
Country of Publication:
United States
Language:
English

References (16)

Construction and characterization of a normalized cDNA library. journal September 1994
Gene–based sequence–tagged–sites (STSs) as the basis for a human gene map journal August 1995
Normalization and subtraction: two approaches to facilitate gene discovery. journal September 1996
3,400 new expressed sequence tags identify diversity of transcripts in human brain journal July 1993
Molecular cloning and nucleotide sequence of cDNA for human liver arginase. journal January 1987
On the Activities of Escherichia coli Exonuclease III journal March 1993
Construction of a uniform-abundance (normalized) cDNA library. journal March 1991
Construction of a normalized cDNA library by introduction of a semi-solid mRNA-cDNA hybridization system journal January 1994
PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. journal March 1994
An ‘equalized cDNA library’ by the reassociation of short double-stranded cDNAs journal January 1990
Rapid cDNA sequencing (expressed sequence tags) from a directionally cloned human infant brain cDNA library journal August 1993
Sequence identification of 2,375 human brain genes journal February 1992
Basic local alignment search tool journal October 1990
Regulation of gene expression: possible role of repetitive sequences journal June 1979
Complementary DNA sequencing: expressed sequence tags and human genome project journal June 1991
Three abundance classes in HeLa cell messenger RNA journal July 1974

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Related Subjects

/435/999/
acid
acid molecule
acid molecules
amplified
appropriate
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cdna
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cdnas
chain
chain reaction
circles
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double-stranded
duplexes
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excising
exonuclease
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full-length cdna
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hybridized
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inserts
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method
methods
molecules
molecules complementary
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normalized
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nucleic
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partial
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polymerase
polymerase chain
primers
produce
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purifying
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separating
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single-stranded
single-stranded dna
single-stranded nucleic
step
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stranded dna
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unhybridized
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