| 摘要 |
The basis of this proposal are the oligonucleotides containing an a-basic group, capable of starting chains. During the amplification reaction (e.g. PCR-polymerase chain reaction) the proposed chain starters are built in to the ends of the amplified nucleic acid fragment 5'-ends. During the amplification reaction the polymerase heading in the direction 3'-5' on the template filament gets stuck at the point where it cannot make sense of the a-basic group. As a result, an amplified product is formed which comprises extended, single strand 5'-ends. These 'sticky' ends make it possible for the direct cloning of the PCR product in the linearised cloning vector without needing any further modification. As far as the amplification reaction is PCR, the method is called 'autosticky PCR'. The proposal also concerns the production of DNA fragments, containing an a-basic group, using oligonucleotides and also methods for the preparation of DNA fragments with 'sticky ends' and methods for the controlled cloning of PCR products having pre-formed sticky ends. The proposed methods can be used in genetic engineering procedures for the preparation of amplified nucleic acid fragments with pre-formed sticky ends e.g. PCR fragments, and the controlled cloning of these fragments. |
