US 5,846,721 A · 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.

Claims30

Dependent claims

A method to normalize a cDNA library comprising:

(a) providing a cloned library containing cDNA inserts in circular double-stranded form wherein the inserts are capable of being amplified by polymerase chain reaction using appropriate primers;
(b) converting the double-stranded cDNA library into single-stranded DNA circles;
(c) generating single-stranded nucleic acid molecules complementary to the inserts of the single-stranded DNA circles converted in step (b) by polymerase chain reaction with appropriate primers and melting double-stranded cDNA inserts produced by the polymerase chain reaction;
(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 at an appropriate Cot;
and
(e) separating unhybridized single-stranded DNA circles from hybridized DNA circles, thereby generating a normalized cDNA library.

3 more elements

Dependencies are inferred from the claim text.

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

Life of this patent

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

Life of this patent

Abstract

Claims30