Amplification of alpha-fetoprotein complementary DNA by insertion into a bacterial plasmid. |
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Authors: | M A Innis M M Harpold D L Miller |
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Affiliation: | 1. Roche Institute of Molecular Biology, Nutley, New Jersey 07110 U.S.A.;2. The Rockefeller University, New York, New York 10021 U.S.A. |
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Abstract: | A fragment of the α-fetoprotein (AFP) structural gene was purified and amplified by bacterial cloning techniques. Double-stranded DNAAFP was constructed from a cDNA copy of greater than 95% pure mRNAAFP and inserted into E. coli plasmid pBR322 by poly(dA-dT)-linkers. Chimeric plasmid DNA isolated from transformants of E. coli strain χ1776 have been shown to contain α-fetoprotein sequences by hybridization to labeled mRNAAFP. One clone, designated pA5 (chimeric plasmid pBR322 containing a cDNAAFP sequence isolated from clone 5), has been studied in more detail. The inserted sequence of approximately 950 nucleotide pairs was positively identified by a hybridization-translation procedure. Hybridization of [3H]uridine-labeled poly(A)-containing RNA from an AFP-secreting cell line to excess pA5 DNA immobilized on nitrocellulose filters was used to show the selectivity of this probe for detecting expression of the AFP gene. |
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