A polymerase chain reaction-based method for cloning novel members of a gene family using a combination of degenerate and inhibitory primers.
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.
Kunihiro
Sumiko
S
Institute of Molecular and Cell Biology, National Institute of Advanced Industrial Science and Technology, Central 6, 1-1, Higashi, Tsukuba, Ibaraki 305-8566, Japan.
Kawanishi
Yuichi
Y
Sano
Motoaki
M
Naito
Kimitoshi
K
Matsuura
Yukio
Y
Tateno
Yoshio
Y
Gojobori
Takashi
T
Yamagata
Youhei
Y
Abe
Keietsu
K
Machida
Masayuki
M
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
D017931:DNA Primers; D018076:DNA, Complementary; D025221:Phosphofructokinases; D006593:Hexokinase; D005941:Glucokinase; D016282:Aspartic Acid Endopeptidases