Methods and compositions for amplifying DNA clone copy number
US7138267B1 · kind B1 · utility
Assignee
Inventors
Key dates
| Filing date | Apr 4, 2002 |
| Grant date | Nov 21, 2006 |
| Priority date | — |
| Expiry date | Feb 13, 2023 |
Classification
- Technology area (CPC C)Chemistry; Metallurgy
- CPC primaryC12N15/69
- WIPO fieldBiotechnology
- WIPO sectorChemistry
Abstract
A method for retrofitting DNA in a single-copy or high-copy vector, such as a fosmid or BAC, whereby an artificial transposon is used to introduce a conditional multi-copy origin of replication (“ori”) into the DNA in said vector. Following random in vitro or in vivo transposition of the ori-containing transposon into DNA in the single-copy or low-copy vector, the resulting insertion clones are introduced into a special host strain that contains a gene which encodes a polypeptide required for replication from the multi-copy ori. However, since the gene for this polypeptide is expressed from a tightly-regulated inducible promoter, the polypeptide is not expressed in the absence of inducer. On addition of inducer to the culture medium, the host cell synthesizes the polypeptide, which in turn activates replication from the multi-copy ori, thereby increasing the amount of clone DNA synthesized by the cell.
Source: USPTO / EPO open patent data. Objective bibliographic and citation counts.