Engineering polymerases and reaction conditions for modified incorporation properties

• US 20100075332A1
• Filed: 09/04/2009
• Published: 03/25/2010
• Est. Priority Date: 09/05/2008
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA), The Scripps Research Institute
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Bibillo, Arkadiusz, Patel, Pranav, Korlach, Jonas, Bjornson, Keith, Hanes, Jeremiah
• Granted Patent: US 8,530,164 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2527/113 Time
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: C12Q 2537/161 A competitive reaction step
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

Provided are methods for enhanced sequencing of nucleic acid templates. Also provided are reaction conditions that increase branching fractions during polymerization reactions. Also provided are compositions comprising modified recombinant polymerases that exhibit branching fractions that are higher than the branching fractions of the polymerases from which they were derived. Provided are compositions comprising modified recombinant polymerases that exhibit delayed translocation relative to the polymerases from which they were derived. Also provided are compositions comprising modified recombinant polymerases that exhibit increased nucleotide or nucleotide analog residence time at an active site of the polymerase. Provided are methods for generating polymerases with the aforementioned phenotypes and methods of using such polymerases to sequence a DNA template or make a DNA. Also provided are methods and nucleic acid sequencing systems for determining which labeled nucleotide is incorporated at a site during a template-dependent polymerization reaction.

Claims

1. A method of determining which nucleotide is incorporated at a site during a template dependent polymerization reaction, the method comprising:

incorporating the nucleotide into a nucleic acid polymer whereby signal pulses or signatures are generated from branch fraction nonincorporation events and, optionally, actual nucleotide incorporation events, for the site;

monitoring a time course of at least branch fraction signal pulses or signatures produced by the polymerization reaction; and

,

assigning which nucleotide is incorporated at the site, using at least signal pulses or signatures from branch fraction nonincorporation sampling events.

2. The method of claim 1, wherein the method comprises counting or estimating the number of redundant iterative sampling signal pulses per incorporation event, or determining an average number of redundant signal pulses per incorporation event.

3. The method of claim 1, wherein the polymerization reaction is a high branch fraction polymerization reaction.

4. The method of claim 3, wherein the branch fraction is at least about 80% or more.

5. The method of claim 1, wherein the reaction comprises at least one species of metal ion, which metal ion increases the frequency of branch fraction nonincorporation events in the reaction.

6. The method of claim 5, wherein the at least one species of metal ion is selected from Mg⁺⁺, Mn⁺⁺, Zn⁺⁺, Co⁺⁺, Ca⁺⁺, Fe⁺⁺, Cr⁺⁺ and Sr⁺⁺.

7. The method of claim 5, wherein the reaction comprises Mg⁺⁺ and Mn⁺⁺.

8. The method of claim 7, wherein the concentration of Mg⁺⁺ is higher than the concentration of Mn⁺⁺.

9. The method of claim 1, wherein the reaction comprises a Φ

29, B103, GA-1, PZA, Φ

15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, L17, T4 or T7 DNA polymerase, or a modified recombinant DNA polymerase thereof.

10. The method of claim 9, wherein the modified recombinant polymerase exhibits a higher branching fraction as compared to a corresponding wild-type polymerase.

12. The method of claim 1, wherein the branch fraction nonincorporation events comprise iterative sampling of labeled unincorporatable nucleotide analogs.

13. The method of claim 12, wherein actual nucleotide incorporation events comprise incorporation of unlabeled nucleotides.

14. The method of claim 12, wherein actual nucleotide incorporation events comprise incorporation of nucleotides that are differentially labeled as compared to the unincorporatable nucleotides.

15. The method of claim 12, wherein the labeled unincorporatable nucleotide analogs comprise a link between an alpha and beta phosphate group that is not hydrolyzable by a polymerase enzyme.

11. (canceled)

16-26. -26. (canceled)

27. A multi-modal sequencing method, comprising:

performing a first template dependent sequencing reaction in a first mode comprising a first set of reaction conditions;

collecting initial sequencing information produced by the first sequencing reaction;

performing a second sequencing reaction of the template, or a copy thereof, in a second mode comprising a second set of reaction conditions;

collecting additional sequencing information produced by the second sequencing reaction; and

,

compiling the initial and additional sequencing information to provide a sequence of at least a portion of the template.

28. The method of claim 27, wherein the second sequencing reaction is produced by altering one or more reaction conditions of the first sequencing reaction, wherein the initial and additional sequencing information are collected in real time.

29-33. -33. (canceled)

34. A method of determining which of two or more labeled nucleotides is incorporated at a site during a template-dependent polymerization reaction, the method comprising:

incorporating the nucleotide into a nucleic acid polymer produced by the polymerization reaction, whereby signal pulses or signatures are generated;

monitoring the pulses or signatures; and

,

using the presence of multiple pulses corresponding to the nucleotide, or identical molecules thereof, to assign which labeled nucleotide is incorporated at the site.

35. The method of claim 34, wherein the multiple pulses comprise 2 to 20 pulses.

36-39. -39. (canceled)

40. A nucleic acid sequencing system that, during operation of the system, sequences a nucleic acid, the system comprising:

a signal detector, which detector detects at least signal pulses or signatures from branch fraction nonincorporation events during sequencing of a template nucleic acid;

system instructions or software that assign a sequence based upon detection of at least signal pulses or signatures from branch fraction nonincorporation events; and

,

a user output module that displays the sequence to the user.

41-42. -42. (canceled)

43. A composition comprising a modified recombinant nucleic acid polymerase, which modified recombinant polymerase exhibits an altered property selected from:

a) an increased branching fraction during a polymerization by the polymerase;

b) an altered translocation property of the polymerase during a polymerization reaction; and

,

c) a combination of any of (a)-(b),

wherein the altered property is altered as compared to a corresponding wild-type polymerase.

44. The composition of claim 43, wherein the modified recombinant polymerase is a modified recombinant Φ

29, B103, GA-1, PZA, Φ

15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, L17, T4, or T7 polymerase.

45. The composition of claim 43, wherein the modified recombinant polymerase that exhibits an increased branching fraction comprises at least one amino acid substitution or deletion or combination of substitutions or deletions selected from:

N62D and Y454A;

D362S;

Y259H;

F237Y;

L381I;

Y369H;

H461Y;

A377G;

K138Q;

H461D;

A377S;

N62D and K371Q;

V118L; and

K124R;

wherein numbering of the residues is relative to a wild-type Φ

29 polymerase of SEQ ID NO;

3.

69. A method of sequencing a nucleic acid template, the method comprising:

a) providing a reaction mixture comprising;

i) the nucleic acid template;

ii) a replication initiating moiety that complexes with or is integral to the template;

iii) the modified recombinant nucleic acid polymerase of claim 43, wherein the polymerase is capable of replicating at least a portion of the template using the moiety in a template-dependent polymerization reaction; and

iv) one or more nucleotides and/or nucleotide analogs;

b) subjecting the reaction mixture to a polymerization reaction in which the modified recombinant polymerase replicates at least a portion of the template in a template-dependent manner, whereby the one or more nucleotides and/or nucleotide analogs are incorporated into the resulting copy nucleic acid; and

c) identifying a time sequence of incorporation of the one or more nucleotides and/or nucleotide analogs into the resulting copy nucleic acid.

73. A method of making a nucleic acid, the method comprising:

a) providing a reaction mixture comprising;

i) a template,

ii) a replication initiating moiety that complexes with or is integral to the template,

iii) the modified recombinant nucleic acid polymerase of claim 43, wherein the polymerase is capable of replicating at least a portion of the template using the moiety in a template-dependent polymerase reaction, and

iv) one or more nucleotides and/or nucleotide analogs; and

b) reacting the mixture such that the polymerase replicates at least a portion of the template in a template-dependent manner, whereby the one or more nucleotides and/or nucleotide analogs are incorporated into the resulting nucleic acid.

46-68. -68. (canceled)

70-72. -72. (canceled)

74-84. -84. (canceled)