Volume 1

Principles and practice of medical genetics / edited by Alan E.H. Emery, David L. Rimoin ; associate editor, Jeffrey A. Sofaer ; editorial assistant, Isobel Black ; foreword by Victor A. McKusick.

Date:
1990
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Principles and practice of medical genetics / edited by Alan E.H. Emery, David L. Rimoin ; associate editor, Jeffrey A. Sofaer ; editorial assistant, Isobel Black ; foreword by Victor A. McKusick. Source: Wellcome Collection.

    56/976 (page 28)
    PLASMID EUKARYOTIC DNA \ I ANNEALING & LIGATION HYBRIDIZATION WITH RADIOACTIVE PROBE AUTORADIOGRAPHY Fig. 2.14 Recombinant DNA cloning. A set of eukaryotic DNA fragments, one of which contains a gene of interest (hatched rectangle) is added to linearized plasmid DNA, and the mixture is annealed and ligated to produce a series of circularized recombinant plasmid DNA molecules. Bacteria transformed by recombinant plasmids are selected in the presence of tetracycline, and the surviving bacterial colonies are screened with a radioactive mRNA probe to locate the colony containing the eukaryotic gene of interest. at very low frequency, and those bacteria which have incorporated a plasmid can, because of the presence of the antibiotic resistance gene on the plasmid, be identified and selected by their ability to grow in the presence of antibiotics. Each single bacterium with a recombinant plasmid can be grown on agar to produce a separate colony containing millions of identical bacteria, each containing one or more identical copies of the same eukaryotic DNA fragment. This eukaryotic DNA can be easily isolated from the bacterial colony after purification and restriction endonuclease digestion of the amplified recombinant plasmid DNA. The final aspect of cloning involves the identification of those bacterial colonies with •tfTGTGATCTC Destroy G Destroy A + G Destroy T+C Destroy C *TGTGATCT0- *TGTGATCT0- *tgtgatcT— *TGTGAT0- * T GTG AT 0 *T GT GAT *TGTGX— *TGT0 *TGT# *TG/ *T£ *T0 on their host bacteria which would otherwise be killed by the antibiotic. The first step in cloning involves the digestion of the plasmid with a restriction enzyme which cuts the plasmid DNA at a single site, opening the circle but leaving the sequence of genes involved in plasmid replication (R in the figure) and antibiotic resistance (TC, tetracycline resistance) intact. The now linear (open) plasmid DNA molecules are then added to a mixture of eukaryotic DNA fragments, one of which contains the gene of interest. By a variety of enzymatic techniques, the ends of each plasmid are joined (ligated) to the ends of a DNA fragment to generate a series of cir cular recombinant plasmid DNA molecules, each of which contains a different eukaryotic DNA fragment. Bacteria which have been exposed to a solution of calcium chloride will take up the recombinant plasmids Fig. 2.15 Sequencing DNA by the method of Maxam and Gilbert. Single strands of DNA, radioactively labelled at one end, are treated with chemical agents that selectively destroy one or two of the four bases, A, T, G and C, thereby cleaving the strands at the site of those bases. The reaction is controlled so that cleavage occurs at only some of the vulnerable sites, thereby generating a set of fragments of different lengths. The DNA is exposed to four separate reaction conditions: a reaction which breaks at Gs alone, one which breaks at Gs and As, one which breaks at Ts and Cs, and one which breaks at Cs alone. The molecules are separated according to size by gel electrophoresis and are visualized by autoradiography. When the autoradiograms corresponding to the four separate reactions are placed side by side, a ladder-like array of bands is generated, representing all of the successively shorter fragments of the original DNA strand. Since one knows which base or pair of bases was destroyed to produce each fragment, one can start at the bottom and read off the sequence of bases.