US 12,270,028 B2
Methods for seamless nucleic acid assembly
Rebecca Nugent, San Francisco, CA (US); Siyuan Chen, San Mateo, CA (US); Elian Lee, Union City, CA (US); and Nathan Raynard, San Francisco, CA (US)
Assigned to Twist Bioscience Corporation, South San Francisco, CA (US)
Filed by Twist Bioscience Corporation, South San Francisco, CA (US)
Filed on Apr. 13, 2022, as Appl. No. 17/720,210.
Application 17/720,210 is a continuation of application No. 16/879,705, filed on May 20, 2020, granted, now 11,332,740.
Application 16/879,705 is a continuation of application No. 16/006,581, filed on Jun. 12, 2018, granted, now 10,696,965, issued on Jun. 30, 2020.
Claims priority of provisional application 62/663,089, filed on Apr. 26, 2018.
Claims priority of provisional application 62/518,496, filed on Jun. 12, 2017.
Prior Publication US 2022/0325278 A1, Oct. 13, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/10 (2006.01); C12N 9/00 (2006.01); C12N 9/12 (2006.01); C12P 19/34 (2006.01); C12Q 1/6811 (2018.01); C12Q 1/6855 (2018.01); C12Q 1/6869 (2018.01)
CPC C12N 15/1096 (2013.01) [C12N 9/1252 (2013.01); C12N 9/93 (2013.01); C12N 15/1031 (2013.01); C12N 15/1093 (2013.01); C12P 19/34 (2013.01); C12Q 1/6811 (2013.01); C12Q 1/6855 (2013.01); C12Q 1/6869 (2013.01); C12Q 2531/101 (2013.01); C12Y 207/07007 (2013.01); C12Y 301/11002 (2013.01)] 20 Claims
 
1. A method for nucleic acid assembly, comprising:
a. providing a plurality of polynucleotides, wherein each of the polynucleotides do not comprise a terminal region of sequence homology to another polynucleotide of the plurality of polynucleotides; and
b. mixing the plurality of polynucleotides with a 3′ to 5′ exonuclease, a thermostable endonuclease, a high fidelity polymerase, and a thermostable ligase, wherein the plurality of polynucleotides are annealed in a processive predetermined order based on a complementary sequence between adjacent polynucleotides.