Methods and devices for nucleic acids synthesis

Patent metadata

Field	Value
Publication number	US-11084014-B2
Application number	US-201916533892-A
Country	US
Kind code	B2
Filing date	Aug 7, 2019
Priority date	Nov 12, 2010
Publication date	Aug 10, 2021
Grant date	Aug 10, 2021

Abstract

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.

First claim

What is claimed is:

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.

Assignees

• Gen9 Inc

Inventors

• Jacobson Joseph
• Kung Li-Yun A
• Wilson Andrew Kirk
• Ramu Senthil
• Schindler Daniel
• Hudson Michael

Classifications

• C12N15/66

  General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease · CPC title

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• B01J2219/00722

  Nucleotides · CPC title

• B01J2219/00585

  Parallel processes · CPC title

• C12N15/1031

  mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR · CPC title