# US 6,294,336 B1 · Method for analyzing the nucleotide sequence of a polynucleotide by oligonucleotide extension on an array

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- Number: US 6,294,336 B1
- Document ID: US6294336B1
- Status: Granted
- Kind: B1
- Application number: 09/522,494
- Filed: 2000-03-09
- Granted: 2001-09-25
- Earliest claimed: 1996-03-19
- Assignees: Orchid BioSciences, Inc.
- Inventors: Michael T. Boyce-Jacino; Yu-Hui Rogers; Philip Goelet
- Web page: https://patentgrep.com/patent/US6294336B1

## CPC

- C12Q 1/6869: Methods for sequencing
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- 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

The present invention provides a method for determining the nucleotide sequence of a nucleic acid molecule using primer oligonucleotides arrayed on a solid support and doing primer extension. By providing sequence information from both oligonucleotide primers in which the 3′ terminal nucleotide of the oligonucleotide primer is hybridized to the nucleic acid molecule and oligonucleotide primers in which the 3′ terminal nucleotide is not hybridized to the nucleic acid molecule the invention is advantageous in that less primers are needed to fully sequence a nucleic acid molecule. Where the 3′ terminal nucleotide of an oligonucleotide in the array does not hybridize to the nucleic acid molecule, the present invention provides for either not permitting oligonucleotides hybridized to nucleic acid molecules to be extended by polymerase-mediated incorporation of a single chain terminator nucleotide residue onto the 3′ terminus of said hybridized oligonucleotide, or permitting the removal of any non-hybridized nucleotide residues from the 3′ terminus of said hybridized oligonucloetides, so as to form a truncated primer oligonucleotide whose 3′ terminus is hybridized to said nucleic acid molecule. The invention also provides kits that can be used to implement the methods of the invention.

## Claims

### Claim 1

A method for determining the nucleotide sequence of a nucleic acid molecule which comprises the steps of:
(A) arraying a set of nested primer oligonucleotides onto a solid support, each array position containing a different array member having a predetermined sequence;
(B) incubating oligonucleotides of said array in the presence of a preparation of said nucleic acid molecules, a polymerase and at least one chain terminator nucleotide; wherein said incubation is under conditions sufficient to permit DNA hybridization to occur between the oligonucleotides of said incubation and said nucleic acid molecules;
(C)
(1) in the case wherein the 3′ terminal nucleotide of an oligonucleotide is hybridized to said nucleic acid molecule, permitting oligonucleotides hybridized to nucleic acid molecules to be extended by polymerase-mediated incorporation of a single chain terminator nucleotide residue onto the 3′ terminus of said hybridized oligonucleotide, wherein for each hybridized oligonucleotide being so extended, said incorporated nucleotide residue is complementary to the nucleotide residue immediately 5′ to the nucleotide residue of the nucleic acid molecule that is hybridized with that oligonucleotide's 3′ terminal nucleotide residue; then performing step (D);
(2) in the case wherein the 3′ terminal nucleotide of an oligonucleotide is not hybridized to said nucleic acid molecule, either:
(a) not permitting oligonucleotides hybridized to nucleic acid molecules to be extended by polymerase-mediated incorporation of a single chain terminator nucleotide residue onto the 3′ terminus of said hybridized oligonucleotide, or
(b) permitting the removal of any non-hybridized nucleotide residues from the 3′ terminus of said hybridized oligonucleotides, so as to form a truncated primer oligonucleotide whose 3′ terminus is hybridized to said nucleic acid molecule, and then permitting polymerase-mediated incorporation of a single chain terminator nucleotide residue onto the 3′ terminus of said hybridized truncated oligonucleotide, wherein for each hybridized truncated oligonucleotide being so extended, said incorporated nucleotide residue is complementary to the nucleotide residue immediately 5′ to the nucleotide residue of the nucleic acid molecule that is hybridized with that truncated oligonucleotide's 3′ terminal nucleotide residue; then performing step (D);
(D) determining, at each array position at which an oligonucleotide has incorporated a single chain terminator nucleotide residue, the identity of the incorporated chain terminator nucleotide residue; and
(E) determining the nucleotide sequence of said nucleic acid molecule from the determined identity of the incorporated nucleotide of primer oligonucleotides of said array, and known sequence of the oligonucleotide at each array position.

### Claim 2 (depends on 1)

The method of claim 1, wherein each array position contains a primer oligonucleotide that is capable of hybridizing to a region of said nucleic acid molecule.

### Claim 3 (depends on 1)

The method of claim 1, wherein said polymerase is a Thermosequenase class polymerase.

### Claim 4 (depends on 1)

The method of claim 1, wherein said polymerase is a Klenow class polymerase.

### Claim 5 (depends on 1)

The method of claim 1, wherein said array is a random oligonucleotide array.

### Claim 6 (depends on 1)

The method of claim 1, wherein said set of nested primer oligonucleotides comprises oligonucleotide members having all possible permutations of nucleotides over a region of from 1 to 20 bases.

### Claim 7 (depends on 1)

The method of claim 1, wherein said method is conducted in the presence of at least four chain terminator nucleotide species, at least one of which is labeled.

### Claim 8 (depends on 7)

The method of claim 7, wherein all of said chain terminator nucleotide species are labeled, and wherein the label of any such species can be distinguished from the label of any other species present.

### Claim 9 (depends on 1)

The method of claim 1, wherein said nucleic acid molecule is a DNA molecule.

### Claim 10 (depends on 1)

The method of claim 1, wherein said nucleic acid molecule is RNA.

### Claim 11 (depends on 1)

The method of claim 1, wherein said method is performed both on said nucleic acid molecule, and on a complement of said nucleic acid molecule.

### Claim 12 (depends on 9)

The method of claim 9, wherein said DNA is genomic DNA of a human or non-human mammal.

### Claim 13 (depends on 9)

The method of claim 9, wherein said DNA is human genomic DNA.

### Claim 14 (depends on 13)

The method of claim 13, wherein said DNA is suspected to contain a genetic variation associated with a disease, and said method is employed to determine whether said DNA contains said variation.

### Claim 15 (depends on 14)

The method of claim 14, wherein said disease is cancer or cystic fibrosis.

### Claim 16 (depends on 1)

The method of claim 1, wherein said oligonucleotides are immobilized onto said solid support.

### Claim 17 (depends on 16)

The method of claim 16, wherein said support is plastic or glass.

### Claim 18 (depends on 1)

The method of claim 1, wherein said set of nested primer oligonucleotides is a non-random oligonucleotide array.

### Claim 19 (depends on 1)

The method of claim 1, wherein the incubation is conducted in the absence of any non-chain terminator nucleotides.

### Claim 20

A kit for determining the sequence of a nucleic acid molecule which comprises a solid support containing an array of spaced apart receptacles for oligonucleotides, each receptacle containing a different primer oligonucleotide, a polymerase capable of removal of any non-hybridized nucleotide residues from the 3′ terminus of a primer oligonucleotide, and at least one chain terminator nucleotide.

### Claim 21 (depends on 20)

The kit of claim 20, further comprising a polymerase not having exonuclease activity.

### Claim 22 (depends on 20)

The kit of claim 20, wherein each array receptacle additionally contains at least four chain terminator nucleotide species, at least on of which is labeled.

### Claim 23 (depends on 20)

The kit of claim 20, wherein all of said chain terminator nucleotide species are labeled, and wherein the label of any such species can be distinguished from the label of any other species present.

### Claim 24

A method for determining the nucleotide sequence of a nucleic acid molecule which comprises the steps of:
(A) providing an array of nested primer oligonucleotides on a solid support, each array position containing a different array member having a predetermined sequence;
(B) incubating the oligonucleotides of said array in the presence of a preparation of said nucleic acid molecules a polymerase and at least one chain terminator nucleotide; wherein said incubation is under conditions sufficient to permit DNA hybridization to occur between the oligonucleotides of said incubation and said nucleic acid molecule;
(C)
(1) in the case wherein the 3′ terminal nucleotide of an oligonucleotide is hybridized to said nucleic acid molecule, permitting the oligonucleotide hybridized to nucleic acid molecules to be extended by polymerase-mediated incorporation of a single chain terminator nucleotide residue onto the 3′ terminus of said hybridized oligonucleotide, wherein for each hybridized oligonucleotide being so extended, said incorporated nucleotide residue is complementary to the nucleotide residue immediately 5′ to the nucleotide residue of the nucleic acid molecule that is hybridized with that oligonucleotide's 3′ terminal nucleotide residue; then performing step (D);
(2) in the case wherein the 3′ terminal nucleotide of an oligonucleotide is not hybridized to said nucleic acid molecule, either:
(a) not permitting oligonucleotides hybridized to nucleic acid molecules to be extended by polymerase-mediated incorporation of a single chain terminator nucleotide residue onto the 3′ terminus of said hybridized oligonucleotide, or
(b) permitting the removal of any non-hybridized nucleotide residues from the 3′ terminus of said hybridized oligonucloetides, so as to form a truncated primer oligonucleotide whose 3′ terminus is hybridized to said nucleic acid molecule, and then permitting polymerase-mediated incorporation of a single chain terminator nucleotide residue onto the 3′ terminus of said hybridized truncated oligonucleotide, wherein for each hybridized truncated oligonucleotide being so extended, said incorporated nucleotide residue is complementary to the nucleotide residue immediately 5′ to the nucleotide residue of the nucleic acid molecule that is hybridized with that truncated oligonucleotide's 3′ terminal nucleotide residue; then performing step (D);
(D) determining, at each array position at which an oligonucleotide has incorporated a single chain terminator nucleotide residue, the identity of the incorporated chain terminator nucleotide residue; and
(E) determining the nucleotide sequence of said nucleic acid molecule from the determined identity of the incorporated nucleotide of primer oligonucleotides of said array, and known sequence of the oligonucleotide at each array position.

### Claim 25 (depends on 24)

The method of claim 24, wherein each array position has a primer oligonucleotide that is capable of hybridizing to a region of said nucleic acid molecule.

### Claim 26 (depends on 24)

The method of claim 24, wherein said polymerase is a Thermosequenase class polymerase.

### Claim 27 (depends on 24)

The method of claim 24, wherein the polymerase is a Klenow class polymerase.

### Claim 28 (depends on 24)

The method of claim 24, wherein the array is a random oligonucleotide array.

### Claim 29 (depends on 24)

The method of claim 24, wherein the array is a non-random oligonucleotide array.

### Claim 30 (depends on 24)

The method of claim 24, wherein the incubation is conducted in the absence of any non-chain terminator nucleotides.

### Claim 31 (depends on 24)

The method of claim 24, wherein said nucleic acid molecule is DNA.