[Construction and selection of bacterial clones hybridized with the mRNA of a light-chain immunoglobulin]

Deev, S M, Karlyshev, A V and Polianovskiĭ, O L (1982) [Construction and selection of bacterial clones hybridized with the mRNA of a light-chain immunoglobulin]. Genetika, 18(6), pp. 880-887. ISSN (print) 0016-6758

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Abstract

The copy-DNA was synthesized on the mRNA fraction which was isolated from MOPC 21 mice myeloma by reverse transcription. This copy was completed with another chain without adding the exogenous primer, with the aid of the Klenov fragment of DNA polymerase I. After treatment of the double-stranded pin DNA with endonuclease S1, the poly(dA) sequences were built up using terminal deoxynucleotidyl transferase. The building up of the poly(dT) sequences at the DNA 3'-termini of pBR322 plasmid previously treated with BamHI was performed in a similar way. The average length of connecting polynucleotide sequences was 50 nucleotides. After annealing and transformation of the cells with Escherichia coli hybrid plasmids, selection of clones was made for ApRTcS phenotype. Further, we applied a stepwise selection of clones by means of increasing the specificity: colony hybridization, quantitative hybridization of the excess of plasmid DNA with (32P)-cDNA and hybridization of plasmid DNAs with mRNA units which were transferred from electrophoregrams to the diazo-papers. As a result, bacterial clones were selected which contained gene fragments showing the ability to hybridize with the RNA fraction characterized by mRNA mobility of the light-chain immunoglobulin G.

Item Type: Article
Research Area: Biological sciences
Faculty, School or Research Centre: Faculty of Science (until 2011) > School of Life Sciences
Depositing User: Andrey Karlyshev
Date Deposited: 06 Mar 2009 11:46
Last Modified: 06 Mar 2009 11:46
URI: http://eprints.kingston.ac.uk/id/eprint/4470

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