MersV1, Main, Exploration, bibRecord, 003A42

Rapid and efficient cloning of cDNAs encoding Krüppel-like zinc finger proteins by degenerate PCR

Identifieur interne : 003A42 ( Main/Exploration ); précédent : 003A41; suivant : 003A43

Rapid and efficient cloning of cDNAs encoding Krüppel-like zinc finger proteins by degenerate PCR

Auteurs : Yasutoshi Agata [Japon] ; Eishou Matsuda [Japon] ; Akira Shimizu [Japon]

Source :

Gene [ 0378-1119 ] ; 1998.

RBID : ISTEX:D1CD2281DC544FA7EB22458688D617E71D1048AE

English descriptors

Teeft :
- Agata, Amino, Amino acids, Available cells, Bamhi, Bamhi site, Blue colonies, Blue selection, Cdna, Certain tissue, Clone, Clones encoding, Degenerate, Degenerate oligonucleotides, Distinct sets, Ecori, Encoding, Equal loading, Gene, Hybridization, Hybridized, Lacz gene, Large number, Limited number, Mok2, Mok2 gene, Mszf, Nger, Nger domain, Nger domains, Nger gene, Nger gene library, Nger genes, Nger protein, Nger proteins, Nger sequences, Northern blot analysis, Novel zinc, Numerous zinc, Primer, Same blot, Spleen, Spleen cells, Testis, Type zinc, Unstimulated, Unstimulated spleen cells, Zinc.

Abstract

Abstract: 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 5×103 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.

Url:

https://api.istex.fr/ark:/67375/6H6-KP42JT7S-9/fulltext.pdf

DOI: 10.1016/S0378-1119(98)00213-3

Affiliations:

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Le document en format XML

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<front><div type="abstract" xml:lang="en">Abstract: 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 5×103 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.</div>
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Rapid and efficient cloning of cDNAs encoding Krüppel-like zinc finger proteins by degenerate PCR

Rapid and efficient cloning of cDNAs encoding Krüppel-like zinc finger proteins by degenerate PCR

Source :

English descriptors

Abstract

Links toward previous steps (curation, corpus...)

Le document en format XML

Pour manipuler ce document sous Unix (Dilib)

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