title: A polymerase chain reaction-based method for cloning novel members of a gene family using a combination of degenerate and inhibitory primers.
abstract: 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.
authors.affiliation: Institute of Molecular and Cell Biology, National Institute of Advanced Industrial Science and Technology, Central 6, 1-1, Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Sumiko
authors.initials: S
authors.lastname: Kunihiro
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Kawanishi
authors.forename: Motoaki
authors.initials: M
authors.lastname: Sano
authors.forename: Kimitoshi
authors.initials: K
authors.lastname: Naito
authors.forename: Yukio
authors.initials: Y
authors.lastname: Matsuura
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Tateno
authors.forename: Takashi
authors.initials: T
authors.lastname: Gojobori
authors.forename: Youhei
authors.initials: Y
authors.lastname: Yamagata
authors.forename: Keietsu
authors.initials: K
authors.lastname: Abe
authors.forename: Masayuki
authors.initials: M
authors.lastname: Machida
mesh_terms: D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000235:genetics*; D001236:Aspergillus oryzae / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D005784:Gene Amplification; D005800:Genes, Fungal / Q000235:genetics*; D005941:Glucokinase / Q000235:genetics; D006593:Hexokinase / Q000235:genetics; D008969:Molecular Sequence Data; D025221:Phosphofructokinases / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D025221:Phosphofructokinases; D006593:Hexokinase; D005941:Glucokinase; D016282:Aspartic Acid Endopeptidases