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PMID: 6287227 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

High-efficiency cloning of full-length cDNA.

Molecular and cellular biology ·Vol. 2 ·No. 2 ·1982-02-00 ·Pages 161-70

Okayama H, Berg P

Abstract

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(5) plasmid-cDNA recombinants obtained per microgram of rabbit reticulocyte mRNA, about 10% contained a complete alpha- of beta-globin mRNA sequence, and at least 30 to 50%, but very likely more, contained the entire globin coding regions. We 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.

MeSH Terms
Animals Base Sequence Cloning, Molecular/methods DNA/analysis DNA Restriction Enzymes DNA, Recombinant/analysis Escherichia coli Globins/genetics Plasmids RNA, Messenger/analysis Rabbits Transformation, Genetic
Chemicals
DNA, Recombinant RNA, Messenger Globins DNA DNA Restriction Enzymes
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Okayama H
Berg P
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37 references, click to expand
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/mcb.2.2.161-170.1982
Published
1982-02-00
Pages
161-70
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC369769
Subset
IM
Grants
NCI NIH HHS · CA-15513-7 · United States
NIGMS NIH HHS · GM-13235 · United States
External Links
PubMed source DOI: 10.1128/mcb.2.2.161-170.1982 PMC Full Text View journal details
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