Efficient and simpler method to construct normalized cDNA libraries with improved representations of full-length cDNAs
- 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.
- 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
Similar Records
Procedure for normalization of cDNA libraries
Procedure for normalization of cDNA libraries
Related Subjects
simpler
method
construct
normalized
cdna
libraries
improved
representations
full-length
cdnas
provides
normalize
library
comprising
constructing
directionally
cloned
containing
inserts
insert
capable
amplified
polymerase
chain
reaction
converting
double-stranded
single-stranded
dna
circles
generating
nucleic
acid
molecules
complementary
converted
step
appropriate
primers
hybridizing
generated
produce
partial
duplexes
cot
separating
unhybridized
hybridized
excising
purifying
cloning
vectors
digesting
exonuclease
subtractive
following
steps
described
methods
acid molecule
cdna libraries
hybridized dna
stranded dna
produce partial
chain reaction
acid molecules
nucleic acid
polymerase chain
cdna library
single-stranded nucleic
cloning vector
partial duplexes
single-stranded dna
normalized cdna
appropriate cot
molecules complementary
full-length cdna
containing cdna
stranded nucleic
dna insert
/435/999/