[Rapid construction and identification of full-length cDNA library of human glioma tissues].
Di Yi Jun Yi Da Xue Xue Bao
; 23(9): 916-20, 2003 Sep.
Article
en Zh
| MEDLINE
| ID: mdl-13129719
ABSTRACT
OBJECTIVE:
To explore a method for rapid construction of a full-length cDNA library of human glioma tissues using switching mechanism at 5' end of RNA transcript (SMART).METHODS:
The total RNA was extracted from several samples of human glioma tissues and the mRNA was subsequently separated. Multiple mRNA samples were mixed to be used as the template for the first-strand cDNA synthesis. The CDS /3' PCR primer (containing Sfi IB site) was used in the first-strand reaction, and the SMART IV Oligo(dT) (containing Sfi A site) served as the short, extended template at the 5' end of the mRNA. With the above two primers, the primer-extension step generated full-length double-strand cDNA, which was digested by Sfi I restriction enzyme and ligated to the Sfi I A & B -digested lambdaTriplEx2 vector. The ligated vector was then packaged by lambda packaging extract for the final construction of the cDNA library.RESULTS:
The unamplified human glioma cDNA library consisted of 2.4x10(6) independent clones with a recombination rate of 100%. The titer of the amplified cDNA library was 4.5x10(9) pfu/ml, and the average exogenous inserts of the recombinants was 1.2 kb in length.CONCLUSION:
A high-quality full-length cDNA library of human gliomas was constructed successfully, which may facilitate further study of the screening and cloning of new tumor suppressor genes and tissue-specific genes of human glioma.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ADN de Neoplasias
/
Biblioteca de Genes
/
ADN Complementario
/
Glioma
Tipo de estudio:
Diagnostic_studies
Límite:
Humans
Idioma:
Zh
Revista:
Di Yi Jun Yi Da Xue Xue Bao
Asunto de la revista:
MEDICINA
Año:
2003
Tipo del documento:
Article
País de afiliación:
China
Pais de publicación:
CHINA
/
CN
/
REPUBLIC OF CHINA