A polymerase chain reaction-based method for cloning novel members of a gene family using a combination of degenerate and inhibitory primers
| Autor: | Yoshio Tateno, Sumiko Kunihiro, Kimitoshi Naito, Yuichi Kawanishi, Motoaki Sano, Keietsu Abe, Yukio Matsuura, Takashi Gojobori, Masayuki Machida, Youhei Yamagata |
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| Rok vydání: | 2002 |
| Předmět: |
DNA
Complementary Aspergillus oryzae Genes Fungal Molecular Sequence Data Saccharomyces cerevisiae Biology Inhibitory postsynaptic potential Polymerase Chain Reaction law.invention law Hexokinase Glucokinase Genetics Gene family Aspartic Acid Endopeptidases Amino Acid Sequence Cloning Molecular Gene Polymerase chain reaction DNA Primers Cloning Sequence Homology Amino Acid Gene Amplification General Medicine Sequence Analysis DNA biology.organism_classification Molecular biology Yeast Phosphofructokinases Primer (molecular biology) Sequence Alignment |
| Zdroj: | Gene. 289(1-2) |
| ISSN: | 0378-1119 |
| Popis: | We have developed a novel method for cloning gene family members by using a polymerase chain reaction technique. The method is based on the amplification of a broad range of homologous genes in combination with the specific inhibition of already cloned genes. To accomplish this, we designed degenerate primers to highly conserved regions among the gene family members, and inhibitory primers to the divergent region at the 3′-margin of each degenerate primer. The 5′-end of the inhibitory primer, the 3′-end of which was aminated, had 3–4 bases overlapping the 3′-end of the degenerate primer. The potential of this method was demonstrated by the successful cloning of a novel member of the yeast MKC7/YAP3 gene family homologue from a filamentous fungus, Aspergillus oryzae, by inhibiting amplification of an already cloned homologue, opsB. |
| Databáze: | OpenAIRE |
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