Methods for Nucleotide Sequencing and High Fidelity Polynucleotide Synthesis

• US 20130244884A1
• Filed: 05/13/2011
• Published: 09/19/2013
• Est. Priority Date: 05/13/2010
• Status: Active Grant

• Current Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Inventors: Jacobson, Joseph, Schindler, Daniel
• Granted Patent: US 10,240,194 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/121 incorporating both deoxyribonucleotides and ribonucleotides
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

Methods of obtaining sequence information about target polynucleotide having a predefined sequence are disclosed. The methods include sequencing by ligation and sequencing by polymerase.

Claims

1. A method for identifying a target polynucleotide having a predefined sequence, the method comprising:

a. providing a support comprising a plurality of anchor oligonucleotides, wherein each plurality of anchor oligonucleotides comprises terminal region having a predefined sequence complementary to a predetermined region of a target polynucleotide having a predefined sequence;

b. contacting the anchor oligonucleotides with the target polynucleotide under conditions suitable to hybridize the predetermined region of the target polynucleotide to the terminal region of the anchor oligonucleotides;

c. contacting the target polynucleotide with at least one sequencing oligonucleotide probe, wherein each oligonucleotide probe comprises at least one interrogating nucleotide at a first interrogation position and a label;

d. ligating the sequencing oligonucleotide probe to the anchor oligonucleotide; and

e. detecting the hybridization of the sequencing oligonucleotide probe to the target polynucleotide thereby identifying the target polynucleotide having the predefined sequence.

2. The method of claim 1 wherein the interrogating position is at the 3′

end of the sequencing probe and each sequencing probe comprises a different nucleotide sequence at the interrogating position.

3. The method of claim 1 wherein the support comprises a plurality of discrete features and wherein each plurality of different anchor oligonucleotides is immobilized at a distinct discrete feature.

4. The method of claim 1 wherein each plurality of anchor probe has a different sequence and different length.

5. The method of claim 2 wherein the different interrogation positions are consecutive.

6. The method of claim 1 wherein the sequencing oligonucleotide comprises at least three degenerate bases and a unique interrogating nucleotide sequence.

7. The method of claim 1 wherein the sequencing oligonucleotide comprises at least three degenerate bases and two or more interrogating nucleotide sequences.

8. The method of claim 1 wherein the nucleotide interrogation position is at the sequencing oligonucleotide 3′

terminus.

9. The method of claim 1 wherein the label comprises a fluorescence tag.

10. The method of claim 1 wherein each plurality of anchor oligonucleotides has a different length and differs from each other from at least one additional predefined interrogation nucleotide.

11. The method of claim 1 wherein each anchor oligonucleotide comprises at least three degenerate bases and differs from each other from at least one additional predefined nucleotide.

12. The method of claim 1 optionally repeating steps c), d) and e) to identify at least one nucleotide at a second interrogation position.

13. The method of claim 1 wherein the different interrogation positions are not consecutive.

14. The method of claim 13 wherein the different interrogation positions are separated by 2, 3, 4, 5, 6, or 7 nucleotides.

15. The method of claim 1 wherein the target polynucleotide comprises a first primer binding site at its 5′

end and a second primer binding site at its 3′

end.

16. The method of claim 1 wherein the target polynucleotide is first amplified before being hybridized to the plurality of anchor oligonucleotides.

17. The method of claim 1 comprising contacting the target polynucleotide with four sequencing oligonucleotide probes, wherein each oligonucleotide probe comprises a unique nucleotide at a first interrogation position and a different label.

18. The method of claim 1 wherein the ligating step is performed using Tth DNA ligase under condition promoting selective ligation.

19. The method of claim 1 further comprising the step of removing unligated probe after the step of ligating.

20. The method of claim 2 comprising sixteen different sequencing probes, each probe comprising two interrogation positions and wherein each of the sixteen sequencing probe is differently labeled.

21. A method for identifying a target polynucleotide having a predefined sequence, the method comprising:

a. providing a support comprising a plurality of anchor oligonucleotides, wherein each plurality of anchor oligonucleotides comprises a terminal region having a predefined sequence complementary to a predetermined region of a target polynucleotide having a predefined sequence;

b. contacting the anchor oligonucleotides with the target polynucleotide under conditions suitable to hybridize the predetermined region of the target polynucleotide to the terminal region of the anchor oligonucleotides;

c. contacting the target polynucleotide with a plurality of sequencing oligonucleotide probes, wherein each plurality of oligonucleotide probes comprises an interrogating RNA nucleotide at an interrogation position and a label;

d. ligating the plurality of sequencing oligonucleotide probes to the plurality of anchor oligonucleotides;

e. detecting the presence or absence of the label wherein the presence of label is correlated to a polynucleotide sequence having the predefined sequence;

f. cleaving the plurality of sequencing oligonucleotide 3′

to the RNA base;

g. repeating steps c) through f) if the presence of the label is detected, until the target polynucleotide having predefined sequence is identified.

22. The method of claim 21 wherein the RNA base is adjacent to the 3′

end nucleotide of the plurality of sequencing oligonucleotides and cleaving the plurality of sequencing oligonucleotide 3′

to the RNA base such as the anchor oligonucleotides are extended by a single nucleotide.

23. The method of claim 21 wherein the RNA base is two bases upstream of the 3′

end nucleotide of the plurality of sequencing oligonucleotides, and cleaving the plurality of sequencing oligonucleotide 3′

to the RNA base such as the anchor oligonucleotides are extended by a dinucleotide.

24. The method of claim 23 wherein the 3′

end nucleotide is an interrogating nucleotide.

25. A method for identifying a target polynucleotide having a predefined sequence, the method comprising:

a. providing a support comprising a plurality of anchor oligonucleotides, wherein each plurality of anchor oligonucleotides comprises a terminal region having a predefined sequence complementary to a predetermined region of a target polynucleotide having a predefined sequence;

b. contacting the anchor oligonucleotides with the target polynucleotide under conditions suitable to hybridize the predetermined region of the target polynucleotide to the terminal region of the anchor oligonucleotides;

c. contacting the target polynucleotide with a plurality of sequencing oligonucleotide probes, wherein each plurality of oligonucleotide probes comprises a single RNA nucleotide at the 3′

end of the plurality of sequencing oligonucleotide probes and a label;

d. ligating the plurality of sequencing oligonucleotide probes to the plurality of anchor oligonucleotides;

e. detecting the absence of the label wherein the absence of label is correlated to a polynucleotide sequence not having the predefined sequence; and

f. cleaving the plurality of sequencing oligonucleotides 3′

to the RNA base, thereby regenerating the support comprising the plurality of anchor oligonucleotides.

26. A method for identifying a target polynucleotide having a predefined sequence, the method comprising:

a. providing a support comprising a plurality of anchor oligonucleotides, wherein each plurality of anchor oligonucleotides comprises a terminal region having a predefined sequence complementary to a predetermined region of a target polynucleotide having a predefined sequence;

b. contacting the anchor oligonucleotides with the target polynucleotide under conditions suitable to hybridize the predetermined region of the target polynucleotide to the terminal region of the anchor oligonucleotides thereby forming at least one target polynucleotide-anchor oligonucleotide duplex;

c. contacting the at least one target polynucleotide-anchor oligonucleotide duplex with at least one labeled nucleotide;

d. extending the anchor oligonucleotide in a template-dependent manner in presence of a polymerase; and

e. detecting the incorporation of the labeled nucleotide into the at least one target polynucleotide-anchor oligonucleotide duplex.

27. The method of claim 26 optionally repeating steps c), d) and e).

28. The method of claim 26 wherein the at least one target polynucleotide-anchor oligonucleotide duplex is contacted with four different labeled nucleotides and wherein each nucleotide is differently labeled.

29. The method of claim 26 wherein each plurality of anchor oligonucleotides has a different length and differs from each other from at least one additional predefined nucleotide.

30. The method of claim 26 wherein each plurality of anchor oligonucleotides comprises at least three degenerate bases and differs from each other from at least one additional predefined nucleotide.

31. The method of claim 26 wherein the target polynucleotide comprises a first primer binding site at its 5′

end and a second primer binding site at its 3′

end.

32. The method of claim 26 wherein the target polynucleotide is first amplified before being hybridized to the plurality of anchor oligonucleotides.

33. The method of claim 26 wherein the support comprises a plurality of discrete features and wherein each plurality of anchor oligonucleotide is immobilized on a distinct discrete feature.

34. A support comprising a plurality of oligonucleotides, wherein each plurality of oligonucleotides is immobilized at a distinct feature of the support, wherein each plurality of oligonucleotides has a different length and comprises a sequence having a terminal region having a predefined sequence, the predefined sequence being complementary to a predetermined region of a target polynucleotide.