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`MERCK v. GENENTECH
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`GENENTECH 2033
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`
`
`
`
`Molecular
`Cloning
`A LABORATORY MANUAL
`
`All rights reserved
`© 1982 by Cold Spring Harbor Laboratory
`Printed in the United States of America
`Book and cover design by Emily Harste
`
`Front cover: The electron micrograph of
`bacteriophage A particles stained with uranyl
`acetate was digitized and assigned false color
`by computer. Thomas R. Broker, Louise T.
`Chow, and James I. Garrels
`Back cover: E. coli DH1 with fimbriae was
`negatively stained with phosphotungstic acid
`and the electron micrograph was digitized and
`assigned false color by computer. Jeffrey A.
`Engler, Thomas R. Broker, and James I.
`Garrels
`
`Cataloging in Publications data
`
`Maniatis, T.
`Molecular cloning.
`
`(A laboratory manual)
`Bibliography: p.
`Includes index.
`2. Eukaryotic
`1. Molecular cloning.
`cells.
`I. Fritsch, Edward F.
`II. Sambrook,
`Joseph.
`III. Title.
`IV. Series.
`QH442.2.F74
`574.87’3224
`ISBN 0—87969—136—0
`
`81-68891
`AACR2
`
`Researchers using the procedures of this manual do so at their own risk. Cold Spring Harbor
`Laboratory makes no representations or warranties with respect to the material set forth in
`this manual and has no liability in connection with the use of these materials.
`
`Other manuals available from Cold Spring Harbor Laboratory
`Hybridoma Techniques
`Experiments with Normal and Transformed Cells
`Advanced Bacterial Genetics,
`Experiments in Molecular Genetics
`A Manual for Genetic Engineering
`(Strain Kit available)
`(Strain Kit available)
`Methods in Yeast Genetics
`
`All Cold Spring Harbor Laboratory publications are available through booksellers or may be ordered
`directly from Cold Spring Harbor Laboratory, Box 100, Cold Spring Harbor, New York.
`SAN 203-6185
`
`
`
`

`
`PLASMIDS
`
`11
`
`Cioning in Plasmids
`
`In principle, cloning in plasmid vectors is very straightforward. The plas-
`mid DNA is cleaved with a restriction endonuclease and joined in vitro to
`foreign DNA. The resulting recombinant plasmids .are then used to trans-
`form bacteria. In practice, however, the plasmid vector must be carefully
`chosen to minimize the effort required to identify and characterize recombi-
`nants. The major difficulty is to distinguish between plasmids that contain
`p sequences of foreign DNA and vector DNA molecules that have recircular-
`ized without insertion of foreign sequences. Recircularization of the plasmid
`can be limited to some extent by adjusting the concentrations of the foreign
`DNA and vector DNA during the ligation reaction. However, a number of
`procedures, described below, have been developed either to reduce recircu-
`larization of the plasmid still further or to distinguish recombinants from
`nonrecombinants by genetic techniques.
`
`Insertional Inactivation
`
`This method can be used with plasmids that carry two or more antibiotic-
`resistance markers (see Fig. 1.1). The DNA to be inserted and the purified
`plasmid DNA are digested with a restriction enzyme that, in this example,
`
`' BamHI BamHI BamHI
`
`foreign DNA
`
`ill
`yyvvm/v\A/vvvvvv~
`
`BamHI
`
`BamHI
`
`
`
`Tonsformants grow in the
`presence of both Tea‘ and Amp
`r
`
`4’.
`
`Tronsformcmts grow in the
`presence of Amp bul not Te?
`
`rigiiire “M
`
`lnsertional inactivation.
`
`
`
`

`
`12 VECTOR-HOST SYSTEMS
`
`recognizes a unique site located in the plasmid within the tetracycline-
`resistance gene. After ligating the two DNAs at the appropriate concentra-
`tions, the ligation mixture is used to transform, for example, ampicillin-sen~
`sitive E’. coli to ampicillin resistance. Some of the colonies that grow in the
`presence of ampicillin will contain recombinant plasmids; others will con-
`tain plasmid DNA that has recircularized during ligation without insertion
`of foreign DNA. To discriminate between the two kinds of transformants, a
`number of colonies are streaked in identical locations on platescontaining
`ampicillin or tetracycline (see Fig. 1.2). The colonies that survive and grow
`in the presence of tetracycline contain plasmids with an active tetracycline— ‘
`resistance gene; such plasmids are unlikely to carry insertions of foreign
`DNA. The colonies that grow only in the presence of ampicillin contain
`plasmids with inactive tetracycline—resistance genes; these plasmids are
`likely to carry foreign DNA sequences.
`.
`In a few cases, methods have been developed to apply positive selection to
`obtain bacteria that are sensitive to an antibiotic from populations that are
`predominantly resistant. In this way, it is. possible to select for recombinant
`plasmids that carry an inactivated antibiotic-resistance gene as a conse-
`quence of insertion of a foreign DNA sequence. The most useful of these
`
`
`
`plates incubated
`
`Colonies of transformed
`bacteria growing in
`the presence of ampicillin
`
`Individual colonies
`transferred with
`a sterile toothpick
`to media containing
`tetracycline (left) or
`ampicillin (right)
`
`ompicillimresistonl, tetracycline —sensilive
`colonies recovered for further analysis
`
`Screening for insertions of foreign DNA by inactivation of plasmid—borne, antibiotic
`resistance genes.
`
`

`
`PLASMIDS
`
`13
`
`\
`
`systems is that described by Bochner et al. (1980) and Maloy and Nunn
`(1981), who developed- media containing the lipophilic, chelating agents
`fusaric acid or quinaldic acid, which allow the direct positive selection of Tet‘
`clones from a population of Tets and Tet’bacteria. For most strains of E. coli,
`approximately 90% of the colonies obtained on media containing tetracycline
`and fusaric acid were found to be Tet‘ when plated onto media containing
`tetracycline alone. It is therefore possible to select from a population of
`bacteria transformed with pBR322 or pAT153 those cells that carry plas-
`mids with insertions at the BamHI and Sall sites.
`A similar technique has been developed to select for bacteria sensitive to
`paromomycin (Slutsky et al. 1980). This should allow the selection of deriva-
`tives of pMK16 that contain insertions at the Smal or Xholsite (Kahn et al.
`1979).
`Although insertion of foreign DNA sequences within an antibiotic-resist
`ance gene almost always leads to inactivation of that gene, at least one case is
`known where an insertion left the gene in a functional state. Villa-Komaroff
`et al. (1978) found that insertion of a segment of rat preproinsulin cDNA into
`the Pstl site of pBR322 did not inactivate the ampicil1in—resistance gene.
`Presumably, a small piece of foreign DNA had been inserted that did not
`alter the reading-frame of the ampicillin—resistance gene, so that a fusion
`protein was formed which retained ,8-lactamase activity.
`
`Directional Cloning
`
`Most plasmid vectors carry two or more unique restriction enzyme recogni-
`tion sites. For example, the plasmid pBR322 contains single Hindlll and
`BamHI sites (see Fig. 1.3). After cleavage by both enzymes, the larger frag— V
`ment of plasmid DNA can be purified by gel electrophoresis and ligated to a
`segment of foreign DNA containing cohesive ends compatible with those
`I generated by BamHI and Hmdlll. The resulting circular recombinant is
`then used to transform E’. coli to ampicillin resistance. Because of the lack of
`complementarity between the Hindlll and BamHI protruding ends, the
`larger vector fragment cannot circularize efficiently; it therefore transforms
`E. coli very poorly. Therefore, most of the colonies resistant to ampicillin
`contain plasmids that carry foreign DNA segments forming a bridge
`between the Hindlll and BamHI sites. Of course, different combinations of
`enzymes can be used, depending on the locations of restriction sites within
`vector and the segment of foreign DNA.
`
`Phosphatase Treatment of Linear, Plasmid Vector DNA
`
`During ligation, DNA ligase will catalyze the formation of a phosphodiester
`bond between adjacent nucleotides only if one nucleotide contains a 5’-
`phosphate group and the other a 3’—hydr0Xyl group. Recircularization of
`plasmid DNA can therefore be minimized by removingethe 5’ phosphates
`from both ends of the linear DNA with bacterial alkaline phosphatase or calf
`intestinal phosphatase (Seeburg et al. 1977; Ullrich et al. 1977). As a result,
`neither strand of the duplex can form a phosphodiester bond. However, a
`
`
`
`

`
`‘IQ VECTOR-HOST SYSTEMS
`
`ill vvvvvvx/v\/\/\/vvx
`
`HindIE[ BamHI
`
`Hind11I
`
`foreign DNA
`
`plasmid vector
`
`’
`
`;BamHI + Hind]II
`
`Ampr
`
`H +
`B
`
`\
`
`
`
`\
`\
`gel electrophoresis I‘
`
`BamHI + Hindm
`
`H
`
`B B
`
`H
`
` %
`
`Tronsformants plated
`on Ampicillin
`Low efficiency of
`High efficiency of
`transformation
`fransfonngfion
`
`Figure 1.3
`
`Directional cloning.
`
`foreign DNA segment with 5’—terminal phosphates can be ligated efficiently
`to the dephosphorylated plasmid DNA to give an open circular molecule
`containing two nicks (see Fig. 1.4). Because circular DNA (even nicked cir-
`cular DNA) transforms much more efficiently than linear plasmid DNA,
`most of the transformants will contain recombinant plasmids. A protocol for
`phosphatase treatment of plasmid DNA is given on page 133.
`
`Problems lfl Cloning Large DNA Fragments in Plasmids
`
`Finally, the size of the foreign DNA to be inserted can also affect the ratio
`of transformants containing recombinant plasmids to those containing recir-
`cularized vectors. In general, the larger the insertion of foreign DNA, the
`lower the efficiency of transformation. Thus, when cloning large DNA frag-
`ments (> 10 kb), it is especially important to take all possible measures to
`keep the number of recircularized vector molecules to a minimum. Even so,
`the background is relatively high, and it is usually necessary to use an in situ
`hybridization procedure (Grunstein and Hogness 19'?’5; Hanahan and Mesel—
`son 1980) to identify recombinant transforrnants.