| Autor: |
DeMarini, D.J., Creasy, C.L., Lu, Q., Mao, J., Sheardown, S.A., Sathe, G.M., Livi, G.P. |
| Zdroj: |
Biotechniques; March 2001, Vol. 30 Issue: 3 p520-523, 4p |
| Abstrakt: |
We have developed an oligonucleotide-mediated cloning technique based on homologous recombination in Saccharomyces cerevisiaethat allows precise DNA sequences to be transferred independent of restriction enzymes and PCR. In this procedure, linear DNA sequences are targeted to a chosen site in a yeast vector by DNA linkers, which consist of two partially overlapping oligonucleotides. The linkers contain relatively short regions of both yeast vector sequences and insert sequences, which stimulate homologous recombination between the vector and the insert. The linkers can also contain sequences not found in either the vector or the insert (e.g., sequences that encode ribosome binding sites, epitope tags, preferred codons, etc.), thus allowing modification of the transferred DNA. Linkers can be designed such that DNA sequences can be transferred with just two reusable universal oligonucleotides and two gene-specific oligonucleotides. This cloning method, which is performed by cotransforming yeast with linear vector, substrate DNA, and unannealed oligonucleotides, has been termed the yeast-based, oligonucleotide-mediated gap repair technique (YOGRT). |
| Databáze: |
Supplemental Index |
| Externí odkaz: |
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