Methods and devices for nucleic acids synthesis
Methods and apparatus relate to the synthesis of polynucleotides having a predefined sequence on a support. Assembly methods include primer extension to generate overlapping construction oligonucleotides and assembly of the polynucleotides of interest onto an anchor support-bound oligonucleotides. Methods and apparatus for selection of polynucleotides having the predefined sequence and/or length are disclosed.
1. A method of generating a polynucleotide having a predefined sequence, the method comprising:
(a) synthesizing a plurality of support-bound double-stranded polynucleotides, each support-bound double-stranded polynucleotide comprising one or more construction oligonucleotides and a free single-stranded overhang, the plurality of support-bound double-stranded polynucleotide sequences comprising the polynucleotide having the predefined 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 sequence of the terminal construction oligonucleotide N;
(c) hybridizing the stem-loop oligonucleotide to the free overhang of the polynucleotide having the predefined sequence;
(d) ligating the stem-loop oligonucleotide to the free overhang of the polynucleotide having the predefined sequence; and
(e) degrading, using an exonuclease, polynucleotide sequences that comprise a free 3′ or 5′ end, and that do not comprise the terminal construction oligonucleotide N.
2. The method of claim 1 , wherein the step of synthesizing comprises hybridizing a pool of oligonucleotides comprising N pluralities of construction oligonucleotides to a plurality of anchor support-bound single-stranded oligonucleotides, wherein the first plurality of construction oligonucleotides in the pool comprises at its 5′ end a sequence region that is complementary to a sequence region at the 5′ end of the plurality of anchor oligonucleotides, and wherein a plurality of construction oligonucleotides N comprises at its 3′ end a sequence complementary to a sequence region of the plurality of construction oligonucleotides (N−1).
3. The method of claim 1 , further comprising degrading the polynucleotide sequences that do not comprise the terminal oligonucleotide sequence using a single strand specific exonuclease.
4. The method of claim 1 , wherein the exonuclease is a single strand-specific 3′ exonuclease, or a single strand-specific 5′ exonuclease.
5. The method of claim 1 , wherein the stem-loop oligonucleotide comprises a type II restriction site.
6. The method of claim 1 , wherein the method further comprises removing the stem-loop oligonucleotide using a type II restriction endonuclease.
7. The method of claim 1 , wherein the stem-loop oligonucleotide comprises at least one Uracil nucleotide.
8. The method of claim 1 , wherein the method further comprises removing the stem-loop oligonucleotide using a mixture of Uracil DNA glycosylase (UDG) and a DNA glycosylase-lyase Endonuclease VIII.
9. The method of claim 1 , further comprising amplifying the predefined polynucleotide having the predefined sequence.
10. The method of claim 1 , wherein in the step of synthesizing the plurality of support-bound double-stranded polynucleotides is synthesized on a support by polymerase chain extension.
11. The method of claim 1 , further comprising releasing the polynucleotide having the predefined sequence from the support.
12. The method of claim 11 , wherein the polynucleotides are released using a Type II restriction enzyme or a mixture of Uracil DNA glycosylase (UDG) and a DNA glycosylase-lyase Endonuclease VIII.