High-efficiency cloning of full-length cDNA

Journal Article · Mon Feb 01 04:00:00 EST 1982 · Mol. Cell. Biol.; (United States)

DOI:https://doi.org/10.1128/MCB.2.2.161· OSTI ID:5880549

Okayama, H; Berg, P

A widely recognized difficulty of presently used methods for cDNA cloning is obtaining cDNA segments that contain the entire nucleotide sequence of the corresponding mRNA. The cloning procedure described here mitigates this shortcoming. Of the 10/sup 5/ plasmid-cDNA recombinants obtained per ..mu..g of rabbit reticulocyte mRNA, about 10% contained a complete ..cap alpha..- or ..beta..-globin mRNA sequence, and at least 30 to 50%, but very likely more, contained the entire globin coding regions. The authors attribute the high efficiency of cloning full- or nearly full-length cDNA to (I) the fact that the plasmid DNA vector itself serves as the primer for first- and second-strand cDNA synthesis, (II) the lack of any nuclease treatment of the products, and (III) the fact that one of the steps in the procedure results in preferential cloning of recombinants with full-length cDNA's over those with truncated cDNA's.

• Research Organization: Dept. of Biochemistry, Stanford Univ. School of Medicine, Stanford, CA 94305
• OSTI ID: 5880549
• Journal Information: Mol. Cell. Biol.; (United States), Journal Name: Mol. Cell. Biol.; (United States) Vol. 2:2; ISSN MCEBD
• Country of Publication: United States
• Language: English

Related Subjects

550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
BIOLOGICAL MATERIALS
BIOTECHNOLOGY
BLOOD
BLOOD CELLS
BODY FLUIDS
CELL CONSTITUENTS
CLONING
DNA
DNA-CLONING
ENZYME ACTIVITY
ENZYMES
ERYTHROCYTES
ESTERASES
GLOBIN
HYDROLASES
MAMMALS
MATERIALS
MESSENGER-RNA
NUCLEASES
NUCLEIC ACIDS
NUCLEOTIDES
OPTIMIZATION
ORGANIC COMPOUNDS
PHOSPHODIESTERASES
PLASMIDS
PROTEINS
RABBITS
RECOMBINANT DNA
RETICULOCYTES
RNA
VERTEBRATES