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

Soares, Marcelo Bento; Bonaldo, Maria de Fatima

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

Patent · Thu Jan 01 00:00:00 EST 1998

OSTI ID:872025

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

New York, NY

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.

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Research Organization:: Columbia Univ., New York, NY (United States)

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)

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

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References (16)

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