Tailored multi-site combinatorial assembly
US9476078B2 · kind B2 · utility
Assignee
Inventor
Key dates
| Filing date | Jul 31, 2008 |
| Grant date | Oct 25, 2016 |
| Priority date | — |
| Expiry date | Feb 23, 2030 |
Classification
- Technology area (CPC C)Chemistry; Metallurgy
- CPC primaryC12N15/1024
- WIPO fieldBiotechnology
- WIPO sectorChemistry
Abstract
The present invention provides a novel method of producing a plurality of modified polynucleotides having different combinations of various mutations at multiple sites by a tailored multi-site combinatorial assembly, comprising adding at least two or at least three primers to a double stranded template polynucleotide in a single reaction mixture, wherein the primers are not overlapping, and wherein each of the primers comprise at least one mutation different from the other primers, wherein at least one primer is a forward primer that can anneal to a minus strand of the template and at least one primer is a reverse primer that can anneal to a plus strand of the template, and subjecting the reaction mixture to a polymerase extension reaction to yield a plurality of extended modified polynucleotides from the at least three primers. The method can be performed without employing a ligation step prior to transforming the extended modified polynucleotides into a cell. The plurality of extended modified polynucleotides can be treated with an enzyme for destroying the template polynucleotide prior to transforming in to the cell.
Source: USPTO / EPO open patent data. Objective bibliographic and citation counts.