# US 5,707,808 A · Optical selection and collection of DNA fragments

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- Number: US 5,707,808 A
- Document ID: US5707808A
- Status: Granted
- Kind: A
- Application number: 08/632,743
- Filed: 1996-04-15
- Granted: 1998-01-13
- Earliest claimed: 1996-04-15
- Assignees: The Regents of the University of California
- Inventors: Mary C. Roslaniec; John C. Martin; James H. Jett; L. Scott Cram
- Web page: https://patentgrep.com/patent/US5707808A

## CPC

- G01N 15/1459: {the analysis being performed on a sample stream}
- C12N 15/1006: {by means of a solid support carrier, e.g. particles, polymers}
- C12Q 1/68: involving nucleic acids
- G01N 15/149: specially adapted for sorting particles, e.g. by their size or optical properties
- G01N 2015/0038: {Investigating nanoparticles}
- Y10T 436/143333: Saccharide [e.g., DNA, etc.]
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- 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
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

Optical selection and collection of DNA fragments. The present invention includes the optical selection and collection of large (>.mu.g) quantities of clonable, chromosome-specific DNA from a sample of chromosomes. Chromosome selection is based on selective, irreversible photoinactivation of unwanted chromosomal DNA. Although more general procedures may be envisioned, the invention is demonstrated by processing chromosomes in a conventional flow cytometry apparatus, but where no droplets are generated. All chromosomes in the sample are first stained with at least one fluorescent analytic dye and bonded to a photochemically active species which can render chromosomal DNA unclonable if activated. After passing through analyzing light beam(s), unwanted chromosomes are irradiated using light which is absorbed by the photochemically active species, thereby causing photoinactivation. As desired chromosomes pass this photoinactivation point, the inactivating light source is deflected by an optical modulator; hence, desired chromosomes are not photoinactivated and remain clonable. The selection and photoinactivation processes take place on a microsecond timescale. By eliminating droplet formation, chromosome selection rates 50 times greater than those possible with conventional chromosome sorters may be obtained. Thus, usable quantities of clonable DNA from any source thereof may be collected.

## Claims

### Claim 1

A method for separating a first group of DNA fragments from a second group of DNA fragments in a mixture thereof, comprising the steps of:
a. preparing a suspension of the DNA fragments in the mixture thereof;
b. staining each DNA fragment in the suspension with at least one analytic fluorescent dye;
c. binding at least one photoinactivating molecule to each DNA fragment in the suspension;
d. hydrodynamically focusing the suspension of stained and bound DNA fragments within the laminar flow field of the sheath fluid in a flow cytometer;
e. permitting individual DNA fragments to pass through at least one beam of light selected to excite the at least one analytic fluorescent dye, thereby producing fluorescence emission from the at least one analytic fluorescent dye thereon;
f. measuring the intensity of the fluorescence emission from the at least one analytic fluorescent dye thereon, whereby individual DNA fragments from the first group and the DNA fragments from the second group are identified;
g. exposing DNA fragments from the second group to light which is absorbed by the at least one photoinactivating molecule bound thereto, whereby DNA fragments from the second group are inactivated and cannot be cloned;
h. collecting DNA fragments from both the first group of DNA fragments and the inactivated DNA fragments from the second group thereof; and
i. cloning the collected DNA fragments, whereby only the first group of DNA fragments will be cloned and thus are separated from the second group of DNA fragments.

### Claim 2 (depends on 1)

The method for separating a first group of DNA fragments from a second group of DNA fragments in a mixture thereof as described in claim 1, wherein the first group of DNA fragments and second group of DNA fragments are derived from chromosomes before said step of preparing a suspension of the DNA fragments in the mixture thereof.

### Claim 3 (depends on 1)

The method for separating a first group of DNA fragments from a second group of DNA fragments in a mixture thereof as described in claim 1, wherein said step of binding at least one photoinactivating molecule to each DNA fragment in the suspension occurs before said step of staining each DNA fragment in the suspension with at least one analytic fluorescent dye.

### Claim 4 (depends on 1)

The method for separating a first group of DNA fragments from a second group of DNA fragments in a mixture thereof as described in claim 1, wherein said at least one fluorescent analytic dye is chosen from the group consisting of Hoechst 33258 and chromomycin A.sub.3.

### Claim 5 (depends on 1)

The method for separating a first group of DNA fragments from a second group of DNA fragments in a mixture thereof as described in claim 1, wherein the at least one photoinactivating molecule is an intercalator or bis-intercalator.

### Claim 6 (depends on 1)

The method for separating a first group of DNA fragments from a second group of DNA fragments in a mixture thereof as described in claim 1, wherein the at least one beam of light is generated from light sources selected from the group consisting of lasers and arc lamps.

### Claim 7 (depends on 1)

The method for separating a first group of DNA fragments from a second group of DNA fragments in a mixture thereof as described in claim 1, wherein each DNA fragment is stained with two analytic fluorescent dyes, and two spaced-apart beams of light are employed to excite the two analytic fluorescent dyes, producing thereby fluorescence emission from the two analytic fluorescent dyes.

### Claim 8 (depends on 1)

The method for separating a first group of DNA fragments from a second group of DNA fragments in a mixture thereof as described in claim 1, wherein said step of exposing DNA fragments from the second group to light which is absorbed by the at least one photoinactivating molecule, bound thereto is achieved using light generated from light sources selected from the group consisting of lasers and arc lamps.

### Claim 9 (depends on 5)

The method for separating a first group of DNA fragments from a second group of DNA fragments in a mixture thereof as described in claim 5, wherein the intercalator is selected from the group consisting of 4,5',8 trimethylpsoralen, 5-aminomethyl-8-methoxypsoralen, and other psoralen derivatives.

### Claim 10 (depends on 5)

The method for separating a first group of DNA fragments from a second group of DNA fragments in a mixture thereof as described in claim 5, wherein the bis-intercalators are selected from the group consisting of TOTO-1, YOYO-1, and other cyanine bis-intercalating dimers.

### Claim 11 (depends on 7)

The method for separating a first group of DNA fragments from a second group of DNA fragments in a mixture thereof as described in claim 7, wherein the two spaced-apart beams of light are generated from light sources selected from the group consisting of lasers and arc lamps.

### Claim 12

A method for separating a first group of DNA fragments from a second group of DNA fragments in a mixture thereof, comprising the steps of:
a. preparing a suspension of the DNA fragments in the mixture;
b. staining each DNA fragment in the suspension with at least one analytic fluorescent dye;
c. binding at least one photoinactivating molecule to each DNA fragment in the suspension;
d. exposing individual DNA fragments to at least one beam of light selected to excite the at least one analytic fluorescent dye, producing thereby fluorescence emission from the at least one analytic fluorescent dye thereon;
e. measuring the intensity of the fluorescence emission from the at least one analytic fluorescent dye thereon, whereby individual DNA fragments are identified as being from the first group of DNA fragments or from the second group of DNA fragments;
f. exposing DNA fragments from the second group to light which is absorbed by the at least one photoinactivating molecule bound thereto, whereby DNA fragments from the second group of DNA fragments are inactivated and cannot be cloned; and
g. cloning the DNA fragments from the first group thereof, whereby the DNA fragments of the second group of DNA fragments are separated from the DNA fragments of the first group.

### Claim 13 (depends on 12)

The method for separating a first group of DNA fragments from a second group of DNA fragments in a mixture thereof as-described in claim 12, wherein the first group of DNA fragments and the second group of DNA fragments are derived from chromosomes before said step of preparing a suspension thereof.

### Claim 14 (depends on 12)

The method for separating a first group of DNA fragments from a second group of DNA fragments in a mixture thereof as described in claim 12, wherein said step of binding at least one photoinactivating molecule to each DNA fragment in the suspension occurs before said step of staining each DNA fragment in the suspension with at least one analytic fluorescent dye.

### Claim 15 (depends on 12)

The method for separating a first group of DNA fragments from a second group of DNA fragments in a mixture thereof as described in claim 12, wherein said at least one fluorescent analytic dye is chosen from the group consisting of Hoechst 33258 and chromomycin A.sub.3.

### Claim 16 (depends on 12)

The method for separating a first group of DNA fragments from a second group of DNA fragments in a mixture thereof as described in claim 12, wherein the at least one photoinactivating molecule is an intercalator or bis-intercalator.

### Claim 17 (depends on 12)

The method for separating a first group of DNA fragments from a second group of DNA fragments in a mixture thereof as described in claim 12, wherein the at least one beam of light is generated from light sources selected from the group consisting of lasers and arc lamps.

### Claim 18 (depends on 12)

The method for separating a first group of DNA fragments from a second group of DNA fragments in a mixture thereof as described in claim 12, wherein each DNA fragment is stained with two analytic fluorescent dyes, and two spaced-apart beams of light are employed to excite the two analytic fluorescent dyes: thereby producing fluorescence emission from the two analytic fluorescent dyes.

### Claim 19 (depends on 12)

The method for separating a first group of DNA fragments from a second group of DNA fragments in a mixture thereof as described in claim 12, wherein said step of exposing DNA fragments from the second group to light which is absorbed by the at least one photoinactivating molecule bound thereto is achieved using light generated from light sources selected from the group consisting of lasers and arc lamps.

### Claim 20 (depends on 16)

The method for separating a first group of DNA fragments from a second group of DNA fragments in a mixture thereof as described in claim 16, wherein the intercalator is selected from the group consisting of 4,5',8 trimethylpsoralen, 5-aminomethyl-8-methoxypsoralen, and other psoralen derivatives.

### Claim 21 (depends on 16)

The method for separating a first group of DNA fragments from a second group of DNA fragments in a mixture thereof as described in claim 16, wherein the bis-intercalators are selected from the group consisting of TOTO-1, YOYO-1, and other cyanine bis-intercalating dimers.

### Claim 22 (depends on 18)

The method for separating a first group of DNA fragments from a second group of DNA fragments in a mixture thereof as described in claim 18, wherein the two spaced-apart beams of light are generated from light sources selected from the group consisting of lasers and arc lamps.