CONFORMATIONAL PROBES AND METHODS FOR SEQUENCING NUCLEIC ACIDS

• US 20110312529A1
• Filed: 06/16/2011
• Published: 12/22/2011
• Est. Priority Date: 06/18/2010
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: HE, MOLLY, CHEN, CHENG-YAO, KOOL, ERIC, RONAGHI, MOSTAFA, PREVITE, MICHAEL, PANTOJA, RIGO
• Granted Patent: US 9,353,412 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2563/125 the label being enzymatic, i.e. proteins, and non proteins, such as nucleic acid with enzymatic activity
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/133 conformational analysis

Abstract

This disclosure provides a method of determining a sequence of nucleotides for a nucleic acid template. The method can include the steps of contacting the nucleic acid template with a conformationally labeled polymerase and at least four different nucleotide species under conditions wherein the conformationally labeled polymerase catalyzes sequential addition of the nucleotide species to form a nucleic acid complement of the nucleic acid template, wherein the sequential addition of each different nucleotide species produces a conformational signal change from the conformationally labeled polymerase and wherein the rate or time duration for the conformational signal change is distinguishable for each different nucleotide species; detecting a series of changes in the signal from the conformationally labeled polymerase under the conditions; and determining the rates or time durations for the changes in the signal, thereby determining the sequence of nucleotides for the nucleic acid template.

Claims

1. A method of determining a sequence of nucleotides for a nucleic acid sample, comprising

providing an array of nucleic acid templates, wherein the nucleic acid templates comprise nucleotide sequence fragments of the nucleic acid sample;

contacting the array of nucleic acid templates with conformationally labeled polymerases and at least four different nucleotide species under conditions wherein the conformationally labeled polymerases catalyze sequential addition of the nucleotide species to form nucleic acid complements of the nucleic acid templates, wherein the different nucleotide species comprise at least one different non-natural nucleotide analog, wherein the sequential addition of each different nucleotide species produces a conformational signal change from the conformationally labeled polymerase and wherein the rate or time duration for the conformational signal change is distinguishable for each different nucleotide species;

detecting a series of changes in the signal from the conformationally labeled polymerase under the conditions and at individual locations of the array; and

determining the rates or time durations for the changes in the signal at the individual locations of the array, thereby determining the sequence of nucleotides for the nucleic acid sample.

2. The method of claim 1, wherein the providing the array comprises amplifying portions of the nucleic acid sample to produce the nucleotide sequence fragments.

3. The method of claim 1, wherein the providing the array comprises adding a universal sequence to the nucleotide sequence fragments to form tailed fragments and hybridizing the tailed fragments to oligonucleotides, wherein the oligonucleotides are attached to the array and wherein the oligonucleotides are complementary to the universal sequence.

4. The method of claim 1, further comprising

contacting the array of nucleic acid templates with conformationally labeled exonucleases under conditions wherein the conformationally labeled exonucleases catalyze sequential removal of nucleotide species from the nucleic acid templates, wherein the sequential removal of each different nucleotide species produces a conformational signal change from each of the conformationally labeled exonucleases and wherein the rate or time duration for the conformational signal change is distinguishable for at least one different nucleotide species that is removed;

detecting a series of changes in the signal from the conformationally labeled exonuclease under the conditions and at the individual locations of the array; and

determining the rates or time durations for the changes in the signal for the series of changes in the signal from the conformationally labeled exonuclease.

5. The method of claim 4, wherein the rate or time duration for the conformational signal change is distinguishable for only one of four different nucleotide species that are removed.

6. The method of claim 4, wherein the rates or time durations for the conformational signal change are distinguishable for four of four different nucleotide species that are removed.

7. The method of claim 4, wherein the at least one different nucleotide species that is removed comprises a modified alpha-phosphate moiety.

8. The method of claim 7, wherein the modified alpha-phosphate moiety comprises a moiety other than oxygen covalently attached to the alpha phosphate.

9. The method of claim 1, wherein the at least one nucleotide species comprise at least four different non-natural nucleotide analogs.

10. The method of claim 1, wherein the conformationally labeled polymerases each comprise an optical probe.

11. The method of claim 10, wherein the conformational signal change comprises a change in wavelength or intensity.

12. The method of claim 10, wherein the optical probe comprises a fluorophore.

13. The method of claim 10, wherein the conformationally labeled polymerases each comprise a pair of optical probes.

14. The method of claim 13, wherein the conformational signal change comprises increased FRET, decreased FRET, increased fluorescence quenching or decreased fluorescence quenching.

15. The method of claim 1, wherein the nucleic acid templates are individually resolved from each other.

16. The method of claim 1, wherein the nucleic acid templates are present in colonies and individual colonies comprise a plurality of templates that are the same species.

17. The method of claim 16, wherein the method comprises determining a sequence of nucleotides for a plurality of templates in the colony.

18. The method of claim 16, wherein the at least one non-natural nucleotide analog comprises a reversible terminator moiety.

19. The method of claim 1, wherein the at least one nucleotide species comprise at least one caged nucleotide.

20. A method of determining nucleotide sequences, comprising

(a) providing an array of different nucleic acid templates;

(b) providing a mixture of nucleotide species, the mixture comprising

(i) at least four different nucleotide species,

(ii) at least one of the four different nucleotide species comprising a reversible terminator moiety, and

(iii) at least two of the four different nucleotide species comprising an extendible 3′

hydroxyl moiety;

(c) contacting the array of nucleic acid templates with conformationally labeled polymerases and the mixture of nucleotide species under conditions wherein the conformationally labeled polymerases catalyze sequential addition of the nucleotide species to form nucleic acid complements of the nucleic acid templates,

wherein the sequential addition of each different nucleotide species produces a conformational signal change from the conformationally labeled polymerase,

wherein the rate or time duration for the conformational signal change is distinguishable for the at least two nucleotide species comprising the extendible 3′

hydroxyl moiety, and

wherein a plurality of the nucleic acid complements incorporate the at least one nucleotide species that comprises the reversible terminator moiety;

(d) removing the reversible terminator moiety;

(e) detecting a series of changes in the signal from the conformationally labeled polymerase at individual locations of the array; and

(f) determining the sequence of nucleotides for the nucleic acid sample from the series of changes in the signal from the conformationally labeled polymerase.

21. The method of claim 20, wherein at least one of the four different nucleotide species is present at a substantially lower concentration than the concentration of any other nucleotide species in the mixture, whereby the nucleotide that is present at the substantially lower concentration produces a distinguishable rate or time duration for the conformational signal change.

22. The method of claim 20, further comprising repeating steps (c), (d) and (e) several times.