US 12,442,028 B2
Method of oligonucleotide synthesis
Michael Chun Hao Chen, Cambridge (GB); and Gordon Ross McInroy, Cambridge (GB)
Assigned to NUCLERA LTD., Cambridge (GB)
Appl. No. 17/436,330
Filed by Nuclera Ltd, Cambridge (GB)
PCT Filed Mar. 9, 2020, PCT No. PCT/GB2020/050559
§ 371(c)(1), (2) Date Sep. 3, 2021,
PCT Pub. No. WO2020/178604, PCT Pub. Date Sep. 10, 2020.
Claims priority of application No. 1903056 (GB), filed on Mar. 7, 2019.
Prior Publication US 2022/0333145 A1, Oct. 20, 2022
Int. Cl. C12P 19/34 (2006.01); C12N 9/12 (2006.01); C12N 9/24 (2006.01); B01J 19/00 (2006.01); C12Q 1/6844 (2018.01); C40B 50/14 (2006.01)
CPC C12P 19/34 (2013.01) [C12N 9/1252 (2013.01); C12N 9/2497 (2013.01); B01J 19/0046 (2013.01); C12Q 1/6844 (2013.01); C40B 50/14 (2013.01)] 20 Claims
 
1. A method for preparing a contiguous oligonucleotide sequence of at least 4n bases in length, the method comprising:
a. taking a device with a plurality of immobilised initiation oligonucleotide sequences, one or more of which contains a cleavage site;
b. using the initiation oligonucleotide sequences to synthesise a plurality of immobilised oligonucleotide sequences of at least n bases in length, using cycles of extension of reversibly blocked nucleotide monomers;
c. selectively cleaving at least four of the immobilised oligonucleotide sequences of at least n bases in length into solution whilst leaving two or more of the immobilised oligonucleotide sequences attached;
d. hybridizing the cleaved and immobilised oligonucleotides to each other, to form splints on at least two of the immobilized oligonucleotide sequences;
e. joining at least one of the cleaved oligonucleotides to at least two of the immobilised oligonucleotide sequences, thereby preparing two separate contiguous oligonucleotide sequences of at least 2n bases in length;
f. performing a further cleavage step of selectively cleaving at least one of the immobilised oligonucleotide sequences of at least 2n bases in length into solution whilst leaving one or more of the immobilised oligonucleotide sequences attached;
g. further hybridizing at least one of the cleaved oligonucleotides to the cleaved oligonucleotide sequences of at least 2n bases, to form a splint on at least one of the immobilized oligonucleotide sequences of at least 2n bases; and
h. further joining at least one of the cleaved oligonucleotides of at least 2n bases to the immobilised oligonucleotide sequences of at least 2n bases, thereby preparing a further contiguous oligonucleotide sequence of at least 4n bases in length.