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US 12,590,324 B2Granted
Compositions and methods for template-free geometric enzymatic nucleic acid synthesis
AssigneeCamena Bioscience Limited
InventorsDerek L. Stemple, Andrew G. Fraser, Sylwia Mankowska, Neil Bell
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Abstract
Disclosed are compositions and methods for template-free nucleic acid synthesis using N-mers and/or anchor primers that comprise at least one XNA or a combination of RNA and DNA.
Claims20
A method comprising:
- a) contacting at least one first plurality of solid supports and at least one first plurality of anchor primers, under conditions that allow for the attachment of a 3′ terminus of at least one anchor primer of the at least one first plurality of anchor primers to at least one solid support of the at least one first plurality of solid supports to produce at least one first anchor primer-substrate complex;
- b) contacting the at least one first anchor primer-substrate complex and at least one first plurality of N-mers under conditions that append a 3′ terminus of at least one N-mer of the at least one first plurality of N-mers to a 5′ terminus of the at least one first anchor primer-substrate to produce at least one first extended anchor primer-substrate complex;
- c) contacting the at least one first extended anchor primer-substrate complex and at least one second plurality of N-mers under conditions that append a 3′ terminus of at least one N-Mer of the at least one second plurality of N-mers to a 5′ terminus of the at least one first extended anchor primer-substrate complex to produce at least one first donor complex;
- d) contacting at least one second plurality of solid supports and at least one second plurality of anchor primers, under conditions that allow for the attachment of a 3′ terminus of at least one anchor primer of the at least one second plurality of anchor primers to at least one solid support of the at least one second plurality of solid supports to produce at least one second anchor primer-substrate complex;
- e) contacting the at least one second anchor primer-substrate complex and at least one third plurality of N-mers under conditions that append a 3′ terminus of at least one N-mer of the at least one third plurality of N-mers to a 5′ terminus of the at least one second anchor primer-substrate to produce at least one second extended anchor primer-substrate complex;
- f) contacting the at least one second extended anchor primer-substrate complex and at least one fourth plurality of N-mers under conditions that append a 3′ terminus of at least one N-mer of the at least one fourth plurality of N-mers to a 5′ terminus of the at least one second extended anchor primer-substrate complex to produce at least one first target complex;
- g) releasing at least one composition comprising the at least one N-mer of the at least one first plurality of N-mers and the at least one N-mer of the at least one second plurality of N-mers from the at least one first donor complex to produce at least one released intermediate complex;
- and
- h) contacting the at least one first target complex and the at least one released intermediate complex under conditions that append a 3′ terminus of the at least one released intermediate complex to a 5′ terminus of the at least one target complex to produce at least one first extended target complex,
- wherein at least one N-mer comprises at least one XNA,
- wherein the anchor primers of the first and second pluralities of anchor primers comprise deoxyribose 5′ (DNA) terminus, and
- wherein at least one of the N-mers comprise a length of 3 to 5 nucleotides and the first, second, third and fourth pluralities of N-mers are chimeric N-mers comprising a deoxyribose 5′ (DNA) terminus and a ribose 3′ (RNA) terminus.
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