Methods for nucleotide sequencing and high fidelity polynucleotide synthesis

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.