Method for in vitro amplification of circular DNA
US6620597B1 · kind B1 · utility
Assignee
Inventors
Key dates
| Filing date | Jul 6, 2000 |
| Grant date | Sep 16, 2003 |
| Priority date | — |
| Expiry date | Jul 6, 2020 |
Classification
- Technology area (CPC C)Chemistry; Metallurgy
- CPC primaryC12Q1/6846
- WIPO fieldBiotechnology
- WIPO sectorChemistry
Abstract
A method for generating and amplifying closed circular DNA having a specific sequence in vitro in a cell-free system is disclosed. Prior to the invention of this method, closed circular DNA could only be amplified in vivo in appropriate host cells. The essence of the method is the inclusion of a thermostable DNA ligase in a PCR reaction. This procedure is referred to as ligation-during-amplification (LDA), in which the fully extended DNA strands are ligated by the DNA ligase and used as templates for subsequent amplification. Closed circular DNA having a specific sequence can be selectively amplified exponentially by the use of two sequence-specific primers in the LDA reaction. In addition, one or more site-specific mutations can be introduced into a closed circular DNA by the use of one or more mutagenic primers in the LDA reaction. Various thermostable DNA polymerases and thermostable ligases can be used for LDA amplification. Any primer position and orientation, either inward or outward, can be used in LDA amplification, as long as there is at least one primer complementary to each strand of the circular DNA. This method has applications in the areas of mutagenesis, cloning, DNA…
Source: USPTO / EPO open patent data. Objective bibliographic and citation counts.