Methods and Devices for Nucleic Acid Synthesis

A method of generating a polynucleotide having a predefined sequence, the method comprising:

• a. synthesizing a plurality of support-bound double-stranded polynucleotides comprising a free single-stranded overhang, the plurality of polynucleotide sequences comprising the predefined polynucleotide sequence, wherein the single-stranded overhang comprises the sequence of a terminal construction oligonucleotide N;
• b. providing a stem-loop oligonucleotide comprising a single-stranded overhang wherein the single-stranded overhang is complementary to the terminal construction oligonucleotide sequence N;
• c. hybridizing the stem-loop oligonucleotide to the free overhang of the polynucleotide having predefined sequence;
• d. ligating the stem-loop oligonucleotide to the free overhang of the polynucleotide having predefined sequence;
• and
• e. degrading polynucleotide sequences that do not comprise the terminal construction oligonucleotide sequence N.

A method of synthesizing a polynucleotide having a predefined sequence, the method comprising:

• a. providing a support with (i) a first plurality of support-bound anchor oligonucleotides wherein the 5′ end of the first plurality of anchor oligonucleotide is complementary to the 5′ end of a first plurality of oligonucleotides and with (ii) a second plurality of support-bound anchor oligonucleotides wherein the 5′ end of the second anchor oligonucleotide is complementary to a terminal construction oligonucleotide N;
• b. synthesizing a plurality of support-bound double-stranded polynucleotides comprising a 5′ single-stranded overhang, the plurality of polynucleotide sequences comprising the predefined polynucleotide sequence, wherein the single-stranded 5′ overhang of the predefined polynucleotide sequence comprises the terminal construction oligonucleotide N sequence and the single stranded 3′ end of the polynucleotide sequence comprises the first oligonucleotide sequence;
• c. hybridizing the plurality of synthesized polynucleotides to the first anchor oligonucleotides;
• and
• d. subjecting the synthesized polynucleotides to hybridization conditions, thereby hybridizing the 5′ overhang of the polynucleotide having terminal construction oligonucleotide N to the 5′ end of the second plurality of anchor oligonucleotides, thereby selecting the polynucleotides having the predefined sequence.

A nucleic acid array comprising:

• a. a solid support;
• b. a plurality of discrete features associated with the solid support wherein each feature comprises a plurality of support-bound oligonucleotides having a predefined sequence wherein the first plurality of oligonucleotides comprises at its 5′ end a sequence region that is complementary to a sequence region at the 5′ end of a second oligonucleotide, wherein a plurality of oligonucleotides N comprises at its 5′ end a sequence complementary to a 5′ end sequence region of a plurality of oligonucleotides (N−1);
• and
• c. at least a first plurality of anchor oligonucleotides comprising at its 5′ end a sequence that is identical to a sequence region of the first plurality of support bound oligonucleotides.

A method for producing a plurality of polynucleotides having a predefined sequence, the method comprising:

• a. providing a first and second supports having a plurality of features, wherein each feature comprises a plurality of different support-bound oligonucleotides having a different predefined sequence;
• b. generating a first and second pluralities of different construction oligonucleotides having different predefined sequences using the plurality of support-bound oligonucleotides as templates, the first and second pluralities of construction oligonucleotides having 3′ end complementary sequences;
• c. providing a support comprising a plurality of features, wherein each feature comprises a plurality support-bound anchor single-stranded oligonucleotides, wherein the 5′ end of each of the plurality of the anchor oligonucleotides is complementary to the 5′ end the first plurality of construction oligonucleotides;
• d. hybridizing the first plurality of construction oligonucleotides to the anchor oligonucleotides;
• e. hybridizing the second plurality of construction oligonucleotides to the first plurality of construction oligonucleotides;
• f. optionally sequentially hybridizing a third plurality of construction oligonucleotides to the second plurality of construction oligonucleotides;
• and
• g. ligating the pluralities of construction oligonucleotides.

A method for synthesizing a plurality of polynucleotides having a predefined sequence, the method comprising:

• a. providing a first support comprising a plurality of features, wherein each feature comprises a plurality support-bound anchor single-stranded oligonucleotides, wherein the 5′ end of each of the plurality of the anchor oligonucleotides is complementary to the 5′ end of a first plurality of construction oligonucleotides;
• b. providing a second support having a plurality of features, wherein each feature comprises a plurality of single-stranded support-bound template oligonucleotides, each plurality of support-bound oligonucleotides having a different predefined sequence;
• c. generating a first plurality of construction oligonucleotides by polymerase extension, using the plurality of support-bound oligonucleotides as templates;
• d. positioning the first and the second support such as each feature of the second support is aligned to a corresponding feature of the first support;
• e. releasing the first plurality of construction oligonucleotides in a solution under conditions promoting hybridization of the first plurality of oligonucleotides to the plurality of corresponding anchor oligonucleotides;
• and
• f. optionally repeating steps b-e with a third support comprising a second plurality of construction oligonucleotides, the second and the third plurality of construction oligonucleotides having 3′ end complementary sequences.