Rapid and efficient cloning of cDNAs encoding Krüppel-like zinc finger proteins by degenerate PCR.
To isolate cDNAs encoding Krüppel-like zinc finger proteins consisting of several hundred members, most of which are yet to be identified, from a limited number of available cells, we developed a rapid and efficient zinc finger gene cloning method based on reverse transcription-polymerase chain reaction (RT-PCR) using tagged, degenerate oligonucleotide primers corresponding to the conserved H/C link followed by the reverse blue selection to identify clones containing properly amplified fragments. More than 5x103 blue colonies were obtained from only 1ng of total RNA. Eighty-eight out of 89 clones, which were randomly picked up from blue colonies and sequenced, encoded 60 different zinc fingers with the expected structure, and among them, only four have been previously described. Furthermore, it was possible to rapidly select clones that were differentially expressed in a tissue and stimulation-specific manner by a differential screening of the zinc-finger cDNA library using probes consisting of distinct sets of the zinc-finger PCR products. These results indicate that our PCR-based method is quite efficient and suitable for analyzing not only zinc finger genes but also other large gene families, especially when the available cells are very limited.
Agata
Y
Y
Center for Molecular Biology, Genetics, Kyoto University, 53 Shougoin-kawaharacho, Sakyo-ku, Kyoto 606-8501, Japan.
Matsuda
E
E
Shimizu
A
A
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics*; D005260:Female; D051379:Mice; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D005810:Multigene Family; D016133:Polymerase Chain Reaction / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012641:Selection, Genetic; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013154:Spleen / Q000166:cytology; D013382:Subtraction Technique; D016335:Zinc Fingers / Q000235:genetics*
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins