Process and reagent for amplifying nucleic acid sequences
US5935825A · kind A · utility
Assignee
Inventors
Key dates
| Filing date | Nov 18, 1994 |
| Grant date | Aug 10, 1999 |
| Priority date | — |
| Expiry date | Nov 18, 2014 |
Classification
- Technology area (CPC Y)Emerging Cross-Sectional Technologies
- CPC primaryY10S435/81
- WIPO fieldBiotechnology
- WIPO sectorChemistry
Abstract
This invention is directed to a novel method for PCR amplification wherein PCR is carried out at a higher pH than the pH widely used in the art. Specifically, the buffer solution is adjusted to pH 9.0 to 11.0 at 25.degree. C. Using the present invention, DNA amplification can be successfully carried out following a simple pretreatment. In the present invention whole blood is mixed with a hypotonic solution so that a selective lysis of red blood cells takes place. The residual leukocytes are then collected. The leukocytes are mixed with a polymerization agent, primers and other necessary reagents and PCR is carried out. When the PCR solution is placed at a high temperature for DNA denaturation, the leukocytes are lysed so that the leukocyte DNA is released and can access the primers and the other necessary reactions for PCR in the solution. Cell membranes and proteins are present in the PCR reaction solution due to the lack of a protein extractive step during the pretreatment. Nevertheless DNA amplification occurs under the presently claimed improved PCR method.
Source: USPTO / EPO open patent data. Objective bibliographic and citation counts.