Mostly natural DNA sequencing by synthesis

• US 8,772,473 B2
• Filed: 03/30/2010
• Issued: 07/08/2014
• Est. Priority Date: 03/30/2009
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Huang, Xiaohua, Roller, Eric
• Primary Examiner(s): Gussow, Anne
• Assistant Examiner(s): Brown, Mindy G
• Application Number:
  US13/262,369
• Publication Number:
  US 20120046177A1
• Time in Patent Office: 1,561 Days
• Field of Search: None
• US Class Current: 536/25.3
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: C12Q 2535/113 Cycle sequencing
• CPC Class Codes: C12Q 2535/119 Double strand sequencing

Abstract

The invention provides a new method for DNA sequencing called “natural sequencing by synthesis” (nSBS). According to the method, DNA that includes a desired sequence is synthesized using a dNTP mix with a small percentage of fluorescently-labeled nucleotides. The fluorescent label is cleavable. In contrast to previous methods that utilize 100% labeled nucleic acids, use of a small percentage of labeled nucleic acids minimizes the distortion of the natural structure of the extending DNA strand and the DNA polymerase. Using the disclosed methods with less than 10,000 copies of template DNA and 10% of the nucleotides labeled, long homopolymer stretches up to 20 bases can be sequenced with high accuracy and Q20 (with 99% accuracy) read lengths of up to 1,000 bases can be achieved. A Q20 read length of greater than 100 bases can potentially be achieved, even if the sequencing is performed with 1,000 copies of a template and 10% of the nucleotides labeled.

Claims

1. A method of determining a target DNA sequence, said method comprising:

a) hybridizing a plurality of polynucleotides that comprise the target DNA sequence with a primer to form a plurality of hybridized templates;

b) contacting the plurality of hybridized templates with i) a DNA polymerase and ii) a first single nucleotide solution, wherein the single nucleotide solution comprises non-terminating, labeled and non-labeled nucleotides, and wherein said percentage of labeled nucleotides incorporated by the DNA polymerase is 20% or less of the total number of nucleotides incorporated, under conditions to allow target DNA-dependent extension from the primer if the appropriate single nucleotide is present;

c) washing to remove unincorporated nucleotides; and

d) detecting the presence or absence of an incorporated, labeled nucleotide among the plurality of hybridized templates, wherein, when the presence of an incorporated, labeled nucleotide is detected, the presence and size, or absence of a stretch of more than one base of the same type in tandem is also detected,

thereby determining the target DNA sequence.

2. The method of claim 1, further comprising:

e) until a signal is detected, repeating steps b)-d) with a second, third, and fourth single nucleotide solution; and

f) optionally cleaving the label from the incorporated labeled nucleotide; and

repeating steps b)-f).

3. The method of claim 1, further comprising:

e) repeating steps b)-d) with a second, third, and fourth single nucleotide solution; and

f) optionally cleaving the label from the incorporated labeled nucleotide; and

repeating steps b)-f).

4. The method according to claim 1, wherein the plurality of hybridized templates are attached to a solid support.

5. The method of claim 4, wherein the solid support comprises more than one distinct area, wherein the plurality of hybridized templates are attached to the solid support in the more than one distinct area.

6. The method of claim 5, wherein the target DNA sequence in each of the plurality of hybridized templates in each distinct area on the solid support is the same.

7. The method of claim 5, wherein the target DNA sequence in each of the plurality of hybridized templates is different between each distinct area on the solid support.

8. The method of claim 5, wherein the method of determining the target DNA sequence is performed in parallel in the more than one distinct areas.

9. The method of claim 1, wherein the percentage of labeled nucleotide incorporated is 1-10% of the total number of nucleotides incorporated.

10. The method of claim 9, wherein the percentage of labeled nucleotide incorporated is 5-10% of the total number of nucleotides incorporated.

11. The method of claim 1, wherein the labeled nucleotides comprise a cleavable linker.

12. The method of claim 1, wherein the target DNA sequence is from genomic DNA.

13. The method of claim 1, wherein the polynucleotide comprising the target DNA sequence is amplified before step a).

14. The method of claim 1, wherein the target DNA sequence is at least 1000 nucleotides in length.

15. The method of claim 1, wherein the primer comprises a hairpin primer that is ligated to the plurality of polynucleotides comprising the target DNA sequence.

16. The method of claim 15, wherein the method further comprises paired end sequencing.