US 12,281,302 B2
Assembly of long nucleic acids by ligation using indexed splint oligos
David Taussig, Sunnyvale, CA (US); and Nicholas M. Sampas, San Jose, CA (US)
Assigned to Agilent Technologies, Inc., Santa Clara, CA (US)
Filed by Agilent Technologies, Inc., Santa Clara, CA (US)
Filed on Aug. 2, 2022, as Appl. No. 17/879,530.
Claims priority of provisional application 63/228,448, filed on Aug. 2, 2021.
Prior Publication US 2023/0037679 A1, Feb. 9, 2023
Int. Cl. C12N 15/10 (2006.01)
CPC C12N 15/1093 (2013.01) 19 Claims
OG exemplary drawing
 
1. A method for assembling two nucleic acid components to produce a multicomponent ligation product, comprising;
(A) hybridizing a first single-stranded oligonucleotide comprising a first DNA sequence component and a second single-stranded oligonucleotide comprising a second DNA sequence component to an indexed splint, wherein both the 5′ end and the 3′ end of the first single stranded oligonucleotide are hybridized to the indexed splint and both the 5′ end and the 3′ end of the second single-stranded oligonucleotide are hybridized to the indexed splint; and
(B) ligating the 3′ end of the first single-stranded oligonucleotide to the 5′ end of the second single-stranded oligonucleotide, thereby producing a first multicomponent ligation product;
wherein the indexed splint comprises from 5′ to 3′:
(a) a second index sequence,
(b) a first spacer sequence which does not hybridize to sequences within the first and the second single-stranded oligonucleotides,
(c) a splint sequence comprising a 5′ element and a 3′ element,
wherein the 5′ element of the splint sequence hybridizes to the 5′ terminal sequence of the second single-stranded oligonucleotide, and
wherein the 3′ element of the splint sequence hybridizes to the 3′ terminal sequence of the first single-stranded oligonucleotide,
(d) a second spacer sequence which does not hybridize to sequences within the first and second single-stranded oligonucleotides, and
(e) a first index sequence.