System and method for nucleic acid sequencing by polymerase synthesis

• US 20030194740A1
• Filed: 05/14/2003
• Published: 10/16/2003
• Est. Priority Date: 12/14/1998
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Li & S Company Limited (Yesco Holdings Co., Ltd.)
• Inventors: Williams, John G.K.
• Granted Patent: US 7,229,799 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C07D 209/12 Radicals substituted by oxygen atoms
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

This invention relates to improved methods for sequencing and genotyping nucleic acid in a single molecule configuration. The method involves single molecule detection of fluorescent labeled PPi moieties released from NTPs as a polymerase extension product is created.

Claims

1. A method of genotyping or sequencing a target nucleic acid comprising the steps of:

i. immobilizing a nucleic acid polymerase or the target nucleic acid onto a solid support in a single molecule configuration;

ii. contacting the solid support with a solution containing;

(a) a target nucleic acid where the polymerase is immobilized or a polymerase where the target nucleic acid is immobilized;

(b) a primer nucleic acid which complements a region of the target nucleic acid downstream of the region to be sequenced;

(c) nucleotide triphosphates [NTP] where each type of base is differently labeled on the phosphate portion, where the labels provide a unique signal that is selectively detectable upon incorporation of NTP into the polymerase extension product;

iii. permitting the polymerase to sequentially extend the primer incorporating the NTP creating a complement to the target nucleic acid; and

,

iv. determining extension of the primer by detecting the unique signal from the labeled NTP to genotype or to sequence the target nucleic acid.

2. A method of claim 1 where the solution contains at least two different types of dNTP.

3. A method of claim 1 where the dNTPs are labeled on the gamma phosphate with a fluorescent label that differentially fluoresces when the gamma phosphate is cleaved away from the nucleoside.

4. A method of claim 1 where the surface of the solid support comprises an array of locations each bearing a single molecule of polymerase or target DNA.

5. A method of claim 1 where the polymerases are immobilized as an array of individual, single molecules onto a solid support.

6. A method of claim 1 where the target DNA is a single nucleotide polymorphism.

7. A method of claim 6 wherein the solution comprises a single dNTP.

8. A method of claim 1 where the detection is a sequential detection of different types of dNTP to provide the sequence of a target DNA.

9. A method of claim 1 wherein the NTP is labeled with a fluorophore on the gamma phosphate and a quencher moiety.

10. A method of claim 1 where the detector is an optical reader.

11. A method of claim 1 where the solution contacting the solid support flows past the immobilized polymerase or target DNA.

12. A method claim 1 wherein the target nucleic acid is deoxynucleotide acid, the polymerase is either a DNA dependent DNA polymerase or a RNA dependent DNA polymerase and the NTPs are dNTPs.

13. A method of claim 1 wherein the target nucleic acid is double stranded DNA and the polymerase is RNA polymerase and the NTPs are rNTPs.

14. A system for genotyping or sequencing a target nucleic acid comprising:

i. a solid support having a surface bearing an immobilized nucleic acid polymerase or the target nucleic acid in a single molecule configuration;

ii. a solution for contacting the surface containing;

(a) a target nucleic acid where the polymerase is immobilized or a polymerase where the target nucleic acid is immobilized;

(b) a molar excess of nucleotide triphosphates [NTP], where each type of NTP base is differently labeled on the phosphate portion and where the labels provide a unique signal that is selectively detectable upon incorporation of the NTP into the polymerase extension product.

15. A system of claim 14 wherein the polymerase is a DNA dependent DNA polymerase, the target nucleic acid is DNA and the NTPs are dNTPs said system further comprising a primer nucleic acid which complements a region of the target nucleic downstream of the region to be sequenced.

16. A system of claim 14 where the surface bears an immobilized DNA dependent DNA polymerase.

17. A system of claim 14 wherein the solution comprises at least two different types of dNTP.

18. A system of claim 14 where the surface of the solid support comprises an array of locations each bearing a single molecule of polymerase or target nucleic acid.

19. A system of claim 14 where the surface of the solid support comprises an array of locations each bearing a single molecule of polymerase.

20. A system of claim 14 where the NTPs are labelled on the gamma phosphate with a fluorescent label that differentially fluoresces when the gamma phosphate is cleaved away from the nucleoside.

21. A system of claim 14 where the target nucleic acid is DNA and where the DNA has a single nucleotide polymorphism from a wildtype DNA.

22. A system of claim 14 wherein the solution comprises a single dNTP.

23. A system of claim 14 wherein the polymerase is a RNA dependent DNA polymerase, the target nucleic acid is RNA, and the NTPs are dNTPs.

24. A system of claim 14 wherein the polymerase is a DNA dependent RNA polymerase, the target nucleic acid is double stranded DNA and the NTPs are rNTPs.

25. A system of claim 14 which further comprises an optical reader.

26. A system of claim of claim 14 which further comprises electrodes where the electrodes are positioned to provide a voltage gradient across the solid support sufficient to permit flow of the solution contacting the immobilized polymerase or target nucleic acid.

28. A solid support of claim 25 wherein the members of the array are addressed.

29. A solid support of claim 25 wherein the polymerase is a DNA dependent DNA polymerase.

30. A solid support of claim 25 wherein the polymerase is a DNA dependent RNA polymerase.

27. A solid support having a surface where the surface has a nucleic acid polymerase array attached to it wherein the members of the array consist of individual molecules of polymerase.