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

United States Patent

5,846,721
December 8, 1998
View the Complete Patent at the US Patent & Trademark Office
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.
Soares; Marcelo Bento (New York, NY), Bonaldo; Maria de Fatima (New York, NY)
The Trustees of Columbia University in the City of New York (New York, NY)
08/ 715,941
September 19, 1996
This invention was made with support under Grant Number DE-FG02-91ER61233 from the U.S. Department of Energy, and Grant Number 1RO1HG00980 from the National Center for Human Genome Research. Accordingly, the U.S. Government has certain rights in the invention.