Benchmarks
(occurring within SwaI) and BamHI restriction sites as well as compatible
non-regenerating BamHI and EcoRI
cohesive ends (Figure 1A). The oligonucleotides were ligated in the presence
of T4 DNA ligase to the SuperCos 1
vector previously digested with BamHI
and dephosphorylated. The DNA that
had been subsequently digested with an
excess of BamHI and fractionated on
agarose gels was purified and then selfligated in the presence of T4 DNA ligase. Following transformation of E. coli
XL1-Blue MR (Stratagene), plasmid
DNA was extracted and analyzed for
the presence of HindIII, PacI, SwaI,
DraI and BamHI restriction sites. The
characteristics of the resulting SuperCos/HPS vector are illustrated in Figure
1B. The vector was subsequently used
for the construction of two cosmid libraries with BamHI or Sau3A partially
digested BHV-1 DNA. Because of the
presence of SwaI and PacI restriction
sites closely flanking the BamHI
cloning site, intact subgenomic inserts
could be readily generated by a simple
digestion of the cosmid recombinants
(not shown). In addition, EcoRI and
HindIII flanking sites could be used to
identify the genomic regions that were
included in the clones by comparing the
restriction patterns obtained to the previously reported restriction maps (1).
Because of its significantly improved
MCS, the SuperCos/HPS vector will facilitate the characterization of large
GC-rich DNA inserts. A similar strategy could be used to introduce GC-rich
restriction sites for the analysis of ATrich DNA inserts.
REFERENCES
1.Mayfield, J.E., P.J. Good, V.H. VanOort,
A.R. Campbell and D.E. Reed. 1983.
Cloning and cleavage site mapping of DNA
from bovine herpesvirus 1 (Cooper strain). J.
Virol. 47:259-264.
2.Plummer, G., C.R. Goodheart, D. Henson
and C.P. Bowling. 1969. A comparative study
of the DNA density and behavior in tissue cultures of fourteen different herpesviruses. Virology 39:134-137.
3.Wahl, G.M., K.A. Lewis, J.C. Ruiz, B.
Rothenberg, J. Zhao and G.A. Evans. 1987.
Cosmid vectors for rapid genomic walking,
restriction mapping, and gene transfer. Proc.
Natl. Acad. Sci. USA 84:2160-2164.
We thank Johnny Basso for his kind assistance in the editing of this manuscript.
814 BioTechniques
This work was funded by the Natural Sciences and Engineering Research Council of
Canada (Grant No. STR0167587). Address
correspondence to Claire Simard, Institut
Armand-Frappier, Centre de recherche en
virologie, 531 Boulevard des Prairies,
Laval des Rapides, QC, Canada H7V 1B7.
Internet: claire_simard@iaf.uquebec.ca
Duplication of a Region in
the Multiple Cloning Site
of a Plasmid Vector to Enhance Cloning-Mediated
Addition of Restriction
Sites to a DNA Fragment
Received 28 May 1997; accepted 1 July
1997.
BioTechniques 23:814-816 (November 1997)
Sirinart Ananvoranich and
Claire Simard
Institut Armand-Frappier
Laval des Rapides, QC, Canada
Cloning a DNA fragment requires
that the ends of the insert be compatible
with the ends of the vector, and maximum efficiency of the ligation is obtained when ends are cohesive. When
adequate cohesive sites are not available, the vector and insert can be rendered blunt by using polymerases and/
or exonucleases, but blunt-end cloning
is technically more difficult to carry out
than cohesive-end cloning, especially
when the insert is bigger than 1 kb. Another approach is to add linkers to the
blunt-ended insert (5). This involves
synthesizing the two oligonucleotides
in the linker, and the resulting DNA
fragment will be delimited by identical
restriction sites after digestion.
An alternative is to clone the fragment using polymerase chain reaction
(PCR) while using primers that contain
restriction sites (7). This method requires knowing the sequence at the
ends of the fragment to be amplified
and synthesizing the two primers; besides, unless special polymerases with
proofreading activity are used in the
PCR (Pfu, Vent, Deep Vent [New
England Biolabs, Beverly, MA, USA]
etc.), this method is restricted to rather
small DNA fragments (<2000 bp).
Another possibility is to clone the
fragment in the multiple cloning site
(MCS) of a vector and then release it
with enzymes that cut at adjacent sites.
A variant of this strategy is connected
with the development of vectors having
a special type of MCS that contains
several couples of restriction sites generating compatible cohesive ends; the
sites in every such couple are separated
by other sites where the insert can be
cloned. One vector of this type is
pSP72 (Promega, Madison, WI, USA),
and its MCS contains the sequence:
XhoI-PvuII-HindIII-SphI-PstI-SalI-...
As SalI and XhoI generate compatible
Vol. 23, No. 5 (1997)
cohesive ends upon cleavage, an insert
can be cloned, for example, in the
HindIII site and can be released by a
SalI + XhoI digestion to be cloned SalI,
XhoI or SalI/XhoI in another vector.
The limitations of this method are the
relatively small number of couples of
sites generating compatible ends, the
number of cloning sites available between any such couple and the fact that
the DNA fragment to be released will
have to be cut away with two different
restriction enzymes, entailing on the
one hand the requirement that both
sites be absent on it, and on the other
hand the impossibility to release the insert from the next cloning vector by a
single digestion for screening purposes.
The new method described here
takes advantage of the special features
of this type of vector and circumvents
the limitations of the previous ones. It
involves rearranging the MCS of the
pSP72/73 plasmids (or any other MCS
that contains couples of sites generating compatible ends). A fragment containing one or a few sites is excised
from the MCS of one copy of the plas-
mid by cutting with two restriction enzymes and is subsequently duplicated
by insertion elsewhere in the MCS of
another copy of the same plasmid; thus,
the new MCS of the latter copy of the
plasmid will contain two fragments that
comprise the same sites.
The principle of the method is illustrated through the example in Figure
1a. Here, fragment AccI-PvuII on the
“acceptor” DNA is replaced by fragment ClaI-SmaI, which contains the
site SacI, from the “donor” DNA; thus,
the site SacI will eventually be present
both 5′ and 3′ to the fragment to be
cloned in site XbaI. For the mentioned
replacement to be possible, the ends of
the fragments generated by restriction
enzyme digestions must be compatible
two by two. In this example duplication, ClaI is compatible with AccI and
SmaI with PvuII. To generalize, the
sites are denoted with X1, X2, Y1, Y2,
C and D symbols on the right side of
Figure 1a, where C is the cloning site
and D is the site to be duplicated. The
configuration of the sites in Figure 1a
(i.e., their order 5′→3′ in the MCS) is
Figure 1. (a) An example illustrating the principle of the method, replacing fragment AccI-PvuII with
fragment ClaI-SmaI. The new MCS contains two SacI sites, so that an insert cloned in XbaI can be released by a SacI digestion. A symbolic notation for the enzymes is provided on the right side to allow
reference to the configurations in b–e. (b) All sites are distinct, and fragments X1-X2 and Y1-Y2 have no
sites in common. (c) X1-X2 and Y1-Y2 are adjacent, and c is the site separating them. (d) No Y1-Y2
fragment is excised from the MCS; instead, X1-X2 is cloned in one site (Y1 = Y2) beyond the cloning
site C (there are three sites in the MCS that generate blunt ends). (e) X1-X2 and Y1-Y2 share a segment
that contains site C. (f) The insert to be cloned in site XbaI (not shown) can be released by digestion with
a single enzyme, SacI, or with two enzymes, SacI and BamHI, both of which were initially located only
5′ to the cloning site.
Vol. 23, No. 5 (1997)
Benchmarks
the one depicted in Figure 1b. Different
configurations for enzymes other than
those considered in the example are
also possible (Figure 1c, d and e). The
cloning site C can be located between
the two MCS fragments (Figure 1b and
d), contained in both (Figure 1e) or, if
they are contiguous, the site that separates them (Figure 1c).
For the particular example above, the
pSP73 plasmid is digested with restriction enzymes to produce the ClaI-SmaI
and AccI-PvuII fragments in two separate reactions. DNA digested with enzymes ClaI and SmaI is also completely
digested with ScaI. The DNA digested
with enzymes AccI and PvuII is then
dephosphorylated (not shown). This has
two consequences: (i) the AccI-PvuII
fragment will not be able to participate
in a ligation reaction with its own vector
if ligase is added, and (ii) the vector
alone will not be able to circularize. In
the next step, aliquots from the two reactions are mixed together in a ligation
reaction. The only ligation that can generate a circular product is that involving
the acceptor dephosphorylated vector
and the ClaI-SmaI fragment. The donor
vector (also digested with ScaI) could,
theoretically, re-ligate the ClaI-SmaI
fragment, but this would be a triplefragment ligation, which is several orders of magnitude less frequent than a
two-fragment ligation. In practice, 1 µg
(about 5 pmol) of plasmid pSP73 is digested with ClaI, SmaI and ScaI restriction enzymes in the donor reaction, and
Figure 2. Screening by restriction enzyme digestion for the loss of a site located in the replaced fragment. The lost site here is PstI for a
configuration where X1 = EcoRV, X2 = XbaI and
Y1 = XbaI, Y2 = PvuII, D = BamHI, C = XbaI.
Lane M: λ DΝΑ/HindIII marker. Lane S: supercoiled pSP73. Lane L: wild-type plasmid linearized with PstI. Lanes a–l: miniprep DNAs digested with PstI. Plasmids in lanes a, e, g and i–k
have lost PstI and have the expected size.
816 BioTechniques
the same amount of DNA is treated with
AccI and PvuII enzymes and then with
alkaline phosphatase in the acceptor reaction. DNAs are eventually redissolved in 10 µL of TE buffer (10 mM
Tris-HCl, pH 8.0, 1 mM EDTA), and 1
µL of each (about 0.1 pmol) is ligated in
10 µL with T4 DNA ligase. One fortieth
of the ligation product is used to transform electro-competent bacteria. Recombinants can be screened both for
having lost one of the sites in the deleted AccI-PvuII fragment and for having
acquired a new one, e.g., the new SacI
site. The two SacI sites are separated by
a small number of base pairs in the new
MCS, and therefore the latter step is
best achieved by sequencing the recombinant plasmid DNA. Figure 2 depicts
clones screened for a lost site in another
example where miniprep DNA has been
prepared by a protocol (3) that made it
also suitable for sequencing by the
Sanger method (6). Alternatively, in
some cases, a duplicated site can also be
detected by releasing a small fragment
in miniprep plasmid DNA by restriction
enzyme digestion. A 100-bp fragment
can be visualized on a 2.5% agarose gel
by increasing the miniprep medium culture volume to 4 mL in a classical
miniprep protocol (1).
The innovation in the method described here is the custom modification
of the MCS of a plasmid vector to make
possible the release of an insert with a
single enzyme or with two enzymes
that are located on the same side of the
cloning site in the unmodified vector
(Figure 1f). The method is simple
(comprising a few digestions and a ligation), efficient (the frequency of recombinants is high) and rapid (no purification of intermediary products is
necessary). The only significant limitation lies with the impossibility to perform both Y1 and Y2 digestions in the
few cases where these sites are adjacent
(2,4). A complete list of all approximately 1400 valid (X1, X2, Y1, Y2, C
and D) combinations can be obtained
from the author by e-mail.
REFERENCES
1.Birnboim, H.C. and J. Doly. 1979. A rapid
alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res.
7:1513-1523.
2.Crouse, J. and D. Amorese. 1986. Double digestions of the multiple cloning site. Bethesda
Res. Lab. Focus 8:9.
3.Del Sal, G., G. Manfidetti and C. Schneider.
1988. A one-tube plasmid DNA mini-preparation suitable for sequencing. Nucleic Acids
Res. 16:9878.
4.New England Biolabs. 1996/1997 Catalog, p.
238-239. New England Biolabs, Beverly, MA.
5.Sambrook, J., E.F. Fritsch and T. Maniatis.
1989. Molecular Cloning: A Laboratory Manual, 2nd ed. CSH Laboratory Press, Cold
Spring Harbor, NY.
6.Sanger, F., S. Nicklen and A.R. Coulson.
1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA
74:5463-5467.
7.Scharf, S.J. 1990. Cloning with PCR, p. 8491. In M.A. Innis, D.H. Gelfand, J.J. Sninsky
and T.J. White (Eds.), PCR Protocols. Academic Press, San Diego.
The author was supported by fellowships
from La Ligue Nationale Contre le Cancer
and Le Rayon Vert. Address correspondence
to Laurentiu Cocea, Institut National de la
Santé et de la Recherche Médicale, Unité
373, Faculté de Médecine Necker, Université
Descartes, 156 rue de Vaugirard, 75730
Paris Cedex 15, France. Internet: cocea@
infobiogen.fr
Received 18 March 1997; accepted 23
May 1997.
Laurentiu Cocea
INSERM, Unité 373
Université Descartes
Paris, France
Stable DNA-Binding
Yeast Vector Allowing
High-Bait Expression for
Use in the Two-Hybrid
System
BioTechniques 23:816-820 (November 1997)
The two-hybrid system has been developed for studying protein-protein interactions in Saccharomyces cerevisiae
cells (1,6,7). Different plasmids have
been constructed, and the screening of
protein partners can vary according to
the biological tools used (10). The
Vol. 23, No. 5 (1997)