# US 5,702,898 A · Procedure for normalization of cDNA libraries

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- Number: US 5,702,898 A
- Document ID: US5702898A
- Status: Granted
- Kind: A
- Application number: 08/465,857
- Filed: 1995-06-06
- Granted: 1997-12-30
- Earliest claimed: 1995-06-06
- Assignees: The Trustees of Columbia University in the City of New York
- Inventors: Maria DeFatima Bonaldo; Marcelo Bento Soares
- Web page: https://patentgrep.com/patent/US5702898A

## CPC

- C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups}
- C12N 15/1096: {cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR}
- C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

This invention provides a method to normalize a cDNA library constructed in a vector capable of being converted to single-stranded circles and capable of producing complementary nucleic acid molecules to the single-stranded circles comprising: (a) converting the cDNA library in single-stranded circles; (b) generating complementary nucleic acid molecules to the single-stranded circles; (c) hybridizing the single-stranded circles converted in step (a) with complementary nucleic acid molecules of step (b) to produce partial duplexes to an appropriate Cot; (e) separating the unhybridized single-stranded circles from the hybridized single-stranded circles, thereby generating a normalized cDNA library.

## Claims

### Claim 1

A method to normalize a cDNA library constructed in a vector capable of being converted to single-stranded circles and capable of producing complementary nucleic acid molecules to the single-stranded circles comprising:
(a) converting the cDNA library to single-stranded circles;
(b) generating antisense RNA in vitro to the single-stranded circles;
(c) hybridizing the single-stranded circles converted in step (a) with antisense RNA of step (b) to produce partial duplexes to an appropriate Cot; and
(d) purifying the unhybridized single-stranded circles from the hybridized single-stranded circles, thereby generating a normalized cDNA library.

### Claim 2 (depends on 1)

The method of claim 1, wherein the cDNA library is a directional cDNA library.

### Claim 3 (depends on 1)

The method of claim 1, wherein the cDNA library is a randomly-primed cDNA library.

### Claim 4 (depends on 1)

The method of claim 1, wherein the cDNA library is constructed in the pT7T3-Pac vector.

### Claim 5 (depends on 1)

The method of claim 1, wherein the antisense RNA is in excess of the single-stranded circles.

### Claim 6 (depends on 3)

The method of claim 3, wherein the antisense RNA is at least one hundred times in excess of the single stranded circles.

### Claim 7 (depends on 1)

The method of claim 1, wherein the antisense RNA and the single-stranded circles are hybridized to a Cot of about five to fifty.

### Claim 8 (depends on 1)

The method of claim 1, wherein the unhybridized single-stranded circles are purified from the hybridized single stranded circles by hydroxyapatite column chromatography.

### Claim 9 (depends on 1)

The method of claim 1, further comprising introducing the unhybridized single-stranded circles into host cells.

### Claim 10 (depends on 9)

The method of claim 9, further comprising conversion of the unhybridized single-stranded circles into double-stranded DNA circles before the introduction into the hosts.

### Claim 11 (depends on 1)

A normalized cDNA library generated by the method of claim 1.

### Claim 12 (depends on 11)

A library of claim 11, wherein the cDNA is derived from a human placenta.

### Claim 13 (depends on 11)

A library of claim 11, wherein the cDNA is derived from human breast tissues.

### Claim 14 (depends on 11)

A library of claim 11, wherein the cDNA is derived from pineal glands in human brain.

### Claim 15 (depends on 11)

A library of claim 11, wherein the cDNA is derived from human retina tissues.

### Claim 16 (depends on 11)

A library of claim 11, wherein the cDNA is derived from ovarian tumor tissues.