A direct method for cDNA cloning employing translation-released mRNA.
1. A new cloning procedure is described for cDNA synthesis from mRNA released by in vitro translation of polysomes in a cell-free amino acid incorporating system. The usefulness of the method lies in the feasibility of employing nanogram amounts of mRNA. 2. Complementary DNA is synthesized directly in the translation mixture simply by adjusting the concentration of some components and removing ribosomes by boiling and centrifugation. 3. As an example, we report here the construction and characterization of a cDNA clone corresponding to chick alpha(I)procollagen starting from a collagen-synthesizing polysome fraction obtained from chick embryos.
Barardi
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CR
Instituto Ludwig de Pesquisa sobre o Câncer, São Paulo, Brasil.
Santos
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CL
Villa
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LL
Brentani
R R
RR
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D000818:Animals; D002458:Cell Fractionation; D002498:Centrifugation; D002642:Chick Embryo; D003001:Cloning, Molecular / Q000379:methods*; D003094:Collagen / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis*; D066298:In Vitro Techniques; D011132:Polyribosomes / Q000378:metabolism*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D046249:Transfer RNA Aminoacylation
D012333:RNA, Messenger; D003094:Collagen; D004247:DNA; D000604:Amino Acyl-tRNA Synthetases