Methods for nucleotide sequencing and high fidelity polynucleotide synthesis
Methods of obtaining sequence information about target polynucleotide having a predefined sequence are disclosed. The methods include sequencing by ligation and sequencing by polymerase.
1. A method for interrogating at least one nucleotide position of a target polynucleotide, the method comprising:
a. providing a support comprising a plurality of anchor oligonucleotides immobilized thereon, wherein each anchor oligonucleotide comprises a terminal region complementary to a first region of a target polynucleotide, wherein at least two anchor oligonucleotides differ in length from one another by one or more predefined nucleotide, and wherein the plurality of anchor oligonucleotides together comprise a predefined portion of the target polynucleotide to be determined;
b. hybridizing the target polynucleotide to a first anchor oligonucleotide at the first region of the target polynucleotide;
c. contacting the target polynucleotide, hybridized to the first anchor oligonucleotide, with a sequencing oligonucleotide probe, wherein the sequencing oligonucleotide probe comprises an interrogating nucleotide at the 3′ and/or 5′ terminus, a label, and a portion complementary to a second region of the target polynucleotide; wherein the sequencing oligonucleotide probe is provided in a pool comprising at least four differently labeled sequencing probes each having a different interrogating nucleotide;
d. ligating the sequencing oligonucleotide probe with the first anchor oligonucleotide when the interrogating nucleotide forms a base pair with the target polynucleotide at a first interrogated position located between the first region and the second region of the target polynucleotide; and
e. detecting the presence of the label in the ligation product of step (d) thereby identifying the presence of a corresponding nucleotide at the first interrogated position on the target polynucleotide, the corresponding nucleotide being complementary to the interrogating nucleotide on the sequencing oligonucleotide probe.
2. The method of claim 1 wherein in step (a), the support comprises a plurality of features, each feature having a plurality of anchor oligonucleotides immobilized thereon that differ in nucleotide sequence from another feature.
3. The method of claim 1 wherein the sequencing oligonucleotide comprises at least three degenerate bases.
4. The method of claim 1 wherein the sequencing oligonucleotide comprises at least three degenerate bases and two or more interrogating nucleotides.
5. The method of claim 1 wherein the interrogating nucleotide is at the 3′ terminus of the sequencing oligonucleotide probe.
6. The method of claim 1 wherein the label comprises a fluorescence tag.
7. The method of claim 1 wherein the first anchor oligonucleotide comprises at least three degenerate bases.
8. The method of claim 1 further comprising repeating steps c), d) and e) using a second sequencing oligonucleotide probe to identify a second interrogated position.
9. 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.
10. The method of claim 1 further comprising, before step b), amplifying the target polynucleotide.
11. The method of claim 1 comprising, in step c), contacting the target polynucleotide with four sequencing oligonucleotide probes, wherein each oligonucleotide probe comprises a unique nucleotide and a different label.
12. The method of claim 1 wherein the ligating step comprises using Tth DNA ligase under condition promoting selective ligation.
13. The method of claim 1 further comprising removing unligated sequencing oligonucleotide probe after the step of ligating.
14. The method of claim 1 wherein the first anchor oligonucleotide is a single-stranded oligonucleotide.
15. The method of claim 1 wherein the first anchor oligonucleotide has at least 5 nucleotides.
16. The method of claim 1 wherein each different anchor oligonucleotide is at least partially complementary to the target polynucleotide.
17. The method of claim 16 wherein each different anchor oligonucleotide comprises bases selected from the group consisting of (i) one or more bases complementary to the target polynucleotide and two or more degenerate bases, (ii) one or more bases complementary to the target polynucleotide and two or more universal bases, and (iii) one or more bases complementary to the target polynucleotide, two or more degenerate bases and two or more universal bases.
18. The method of claim 1 wherein each different anchor oligonucleotide has a common n-mer core.
19. The method of claim 1 wherein each different anchor oligonucleotide differs from one another by a different terminal base.
20. The method of claim 1 wherein each different anchor oligonucleotide differs from one another such that in totality they comprise complementarity to the predefined sequence of the target polynucleotide.
21. The method of claim 2 wherein the plurality of anchor oligonucleotides are immobilized on the support at their 3′ end.
22. The method of claim 2 wherein the plurality of anchor oligonucleotides are immobilized on the support via a linker.
23. The method of claim 2 wherein the plurality of anchor oligonucleotides are immobilized on the support via a cleavable linker.