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Epicentre Forum 5 (3)
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Mike Fiandt, Epicentre Technologies
Cosmid vectors are high capacity genomic cloning vectors that contain
an origin of replication, a selectable drug resistance marker, and the
cos site of phage lambda. In a typical cosmid cloning experiment, a sized Sau3A
I partial digest of genomic DNA (30-45 kb) is ligated to a cosmid vector
that has been linearized with BamH I. A properly sized segment
of genomic DNA flanked by cosmid molecules with cos sequences in the
same orientation is then packaged using a lambda in vitro packaging system.
Following infection of the appropriate host strain, the cosmid clones
are selected on an antibiotic plate.
There are several problems commonly encountered using this approach.
First, the genomic DNA must be at least 200 kb in length before partial
digestion or only a small fraction of the 30-45 kb molecules will contain
a restriction site at both ends.1 Isolating
quality DNA of this size from some organisms and tissues can be difficult.
Secondly, obtaining satisfactory partial digests of high molecular weight
eukaryotic DNA can be difficult and time consuming. Finally, a Sau3A
I partial digest is not truly random, possibly leading to failure to
yield a particular clone.
To circumvent these problems, we have developed the pWEB™ Cosmid
Cloning Kit. The pWEB Kit utilizes a vector containing a blunt-ended
cloning site (Sma I). Randomly sheared genomic DNA is end-repaired,
sized on a low melting point agarose gel, and subsequently cloned into
the pWEB Vector. In this report, we demonstrate the high efficiencies
obtainable using this approach, and present results for the construction
of both eukaryotic DNA (wheat and mouse) and bacterial DNA libraries.
The pWEB Cosmid Cloning Kit allowed us to construct a high efficiency
(4 x 106 cfu/µg insert DNA) wheat DNA library in two
days.
The pWEB Vector
The pWEB Vector, a derivative of pWE15,2 is
depicted in Figure 1. The vector has a number of features that make it
well suited for use in cosmid cloning. pWEB contains a unique Sma I
site for the cloning of blunt-ended genomic DNA. This site is flanked
by rare cutting Not I sites for the subsequent excision of insert
DNA. The vector contains a col E1 origin of replication and ampicillin
resistance for growth in E. coli, and an SV40 origin of replication
and neomycin resistance for selection in eukaryotic cells. The vector
contains M13 forward and T7 promoter primer binding sites that can be
used for sequencing insert ends and generating probes for chromosome
walking. Restriction mapping of insert DNA can be performed using lambda
terminase and labeled oligonucleotides complementary to the cos sites
(ON-R and ON-L). The pWEB Vector is conveniently supplied as a Sma I-linearized,
dephosphorylated vector.
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Figure 1. Circle map and cloning region of the pWEB Vector
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Control insert
DNA from phage T7 is provided as a control for the pWEB system. At
40 kb in size, the T7 Control DNA cloned into the pWEB Vector results
in a 48 kb molecule, the optimal size for lambda packaging. The blunt-ended
T7 Control DNA is ligated to the pWEB Vector and packaged using MaxPlax™ Lambda
Packaging Extract. When the packaged pWEB/T7 molecule is transfected
into a cell, T7 phage development ensues, resulting in the formation
of T7 plaques, instead of the colonies normally obtained in a cosmid
cloning experiment.
General pWEB procedure
A general outline of the procedure is shown in Figure 2. Because genomic
DNA should be in the 30-45 kb range for optimal cloning efficiency, DNA
greater than 50 kb is first sheared to the correct size range by expelling
the DNA solution from a syringe through a small bore needle. The sheared
DNA is then end-repaired to generate blunt ends for cloning into the
pWEB Vector. The end-repaired DNA is resolved on a low melting point
agarose gel and the DNA in the correct size range is excised. Next, the
insert DNA is purified and concentrated from the gel slice using GELase™ Preparation
and then ligated to the pWEB Vector. Alternatively, the ligation can
be carried out directly in the gel. This in-gel ligation procedure is
faster and more convenient, and in many cases, yields sufficient clones
for a complete library. The ligation products are packaged using MaxPlax
Lambda Packaging Extract, a restriction-free, high-efficiency in vitro packaging
system. EPI305™ E. coli cells (restriction-free and recA-)
are then infected with the phage particles and plated onto LB/ampicillin
plates. Use of restriction-free MaxPlax Extracts and the EPI305 strain
is especially important for packaging of DNA bearing mammalian methylation
patterns.
Wheat DNA library construction using two different ligation procedures
Wheat germ DNA was isolated using standard methods.1 The
DNA was sheared by expelling the DNA from a syringe through a 25 gauge
needle. The DNA was then end-repaired in a reaction containing 1X End-Repair
Buffer, 0.25 mM each dNTP, 1 mM ATP, 20 µg of sheared genomic DNA,
and 4 µl of End-Repair Enzyme Mix in a total volume of 80 µl.
After a 45-minute incubation at room temperature, 16 µl of gel
loading dye were added and the reaction was incubated for 10 minutes
at 70°C. The end-repaired DNA was loaded onto a 1% low melting point
(LMP) agarose preparative gel with 100 ng of T7 DNA (40 kb) in the outside
lanes as a marker. The gel was run overnight at 30 volts at room temperature.
The outside lanes containing the T7 markers were cut from the gel, stained
with ethidium bromide, and the position of the T7 bands marked by inserting
a pipet tip. The gel was reassembled and the genomic DNA migrating between
the marker bands was excised. Alternatively, pulsed field gel electrophoresis
(PFGE) or field inversion gel electrophoresis (FIGE) can be used to allow
for more precise size fractionation.
Ligation was then performed either directly in the gel, or following
purification and concentration of the DNA. The 50 µl in-gel ligation
reaction contained 25 µl of melted 1% LMP gel containing the size-selected
insert DNA, 1X Fast-Link™ Buffer, 1 mM ATP, 2 units of Fast-Link
DNA Ligase, and 0.5 µg of pWEB Vector. The mixture was allowed
to solidify and the ligation was allowed to proceed for two hours at
room temperature. The reaction was then heated at 70°C for 10 minutes
to melt the gel and inactivate the ligase, cooled to ~40-45°C, and
treated with 1 unit of GELase Preparation for 15 minutes. The DNA (10 µl)
was directly packaged using MaxPlax Packaging Extract according to the
protocol3 and dilutions of the extract
were mixed with EPI305 host bacteria and plated onto LB/ampicillin plates.
The plates were incubated overnight at 37°C.
For ligation using purified and concentrated DNA, the excised gel slice
was first incubated for 10 minutes at 70°C to melt the agarose. The
DNA was then treated with 3 units of GELase Preparation for 30 minutes
at 42°C. The sample was incubated for 10 minutes at 70°C to inactivate
the GELase enzyme and the DNA was precipitated with two volumes of 100%
ethanol. Then, 1 µg of pWEB Vector was ligated with the concentrated
wheat DNA for 2 hours in a 10 µl ligation reaction. After heating
for 10 minutes at 70°C, the entire ligation reaction was packaged
using MaxPlax Packaging Extract. Dilutions of the extract were mixed
with EPI305 host bacteria, spread on LB/ampicillin plates, and the plates
were incubated overnight at 37°C.
Construction of a mouse DNA library
Mouse liver DNA was purified from nuclei.4 The
DNA was then sheared, end-repaired, and sized on a 1% low melting point
agarose gel as described above. The ~40 kb DNA was then excised from
the gel, and purified and concentrated using GELase Preparation as described
above. The concentrated DNA was ligated with 1 µg of pWEB Vector
for 2 hours in a 10 µl ligation reaction. The entire ligation reaction
was packaged using MaxPlax Packaging Extract. Dilutions of the extract
were mixed with EPI305 host bacteria, spread on LB/ampicillin plates,
and the plates were incubated overnight at 37°C.
Construction of a bacterial DNA library
Bacterial DNA from Oerskovia xanthineolytica was purified, sheared,
end-repaired, and sized on a 1% low melting point agarose gel as described
above. For ligation, the in-gel procedure was used. The in-gel ligation
contained 25 µl of melted 1% LMP gel containing the size-selected
insert DNA and 0.5 µg of pWEB Vector. Following ligation, the sample
was treated with GELase Preparation and the entire reaction (10 µl)
was packaged using MaxPlax Packaging Extract. Dilutions of the extract
were plated as above.
Control template and background reactions
A control experiment was performed by ligating 0.5 µg of T7 Control
DNA with 1.0 µg of pWEB Vector (vector:insert ratio = 10:1) in
a 10 µl overnight ligation reaction at room temperature. The ligase
was inactivated by heating for 10 minutes at 70°C and the entire
reaction was packaged using MaxPlax Packaging Extract. The packaging
reaction was serially diluted, adsorbed to LE392MP indicator bacteria,
and plated to determine T7 phage titer.
To test for vector background, an in-gel ligation was carried out as
described above, except no insert DNA was added to the ligation. The
self-ligated vector DNA was packaged and plated on EPI305 host cells
as described above.
The results of each experiment are shown in Table 1. The wheat germ
DNA packaging reaction containing DNA that was concentrated prior to
ligation yielded 2.8 x 105 total clones, which corresponds
to 2.6 x 106 clones/µg of insert DNA. These values are
much higher than the values typically reported for eukaryotic cosmid
libraries, which range from 5 x 104 clones/µg DNA to
1 x 106 clones/µg DNA.1,2,5,6 These
high efficiencies demonstrate that in some cases, it may be technically
and economically feasible to construct and screen cosmid libraries without
amplification using the pWEB system. This would be of great benefit since
amplification of cosmid libraries often results in the selective loss
of clones or allows for rearrangements of the genomic DNA.
When the in-gel ligation procedure was used, the packaging reaction
yielded 5 x 104 clones. Using fluorometrically determined
DNA concentrations in the gel slice, the efficiency was calculated to
be 4.5 x 106 cfu/µg of insert DNA. Thus, the efficiency
of cloning was approximately equal for the in-gel ligation procedure
and the procedure using concentrated DNA; however, use of the concentrated
DNA procedure produced more clones per packaging reaction.
DNA minipreps were prepared from several wheat germ clones and analyzed
by restriction digestion with Not I, EcoR I, and BamH
I (Figure 3). The 8.2 kb vector band is clearly visible in all three
digests, and the insert DNA produced the expected variable patterns.
| Figure 3. Restriction analysis of wheat germ
clones |
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A mouse DNA library was produced from DNA that was purified from the
gel and concentrated. The yield was 1.3 x 105 cfu/packaging
extract and the efficiency was calculated to be 5.8 x 105 cfu/µg
insert DNA. Whether the difference in cloning efficiency between wheat
and mouse was due to an intrinsic difference in the DNA or just a reflection
of the particular DNA preparation was not determined.
The yield of cosmid clones from the O. xanthineolytica packaging
reaction was 5 x 104 clones per extract. From fluorometric
measurements of insert DNA concentration in the gel slice, it was calculated
that the yield of cosmid clones was 3.3 x 107 cfu/µg
of insert DNA. Of 100 cosmid clones screened, 3 positives were found
for the gene of interest.
The control experiment yielded 3-6 x 107 pfu/µg T7
DNA (Table 1). These efficiencies demonstrate that blunt-end ligations
are not a limiting factor in this cosmid cloning system. Control experiments
in which the pWEB Vector was omitted resulted in the formation of zero
plaques.
The vector background ligation resulted in the formation of 40 cfu
per packaging extract. This background was at least several orders of
magnitude lower than the number of clones obtained in the wheat germ
DNA cloning experiment.
We have developed a high-efficiency, low-background cosmid cloning
system for library construction. Randomly sheared genomic DNA is end-repaired
to generate blunt ends for ligation into the Sma I pre-digested,
dephosphorylated pWEB Vector. This approach eliminates the difficulties
of isolating very high molecular weight genomic DNA and generating partial
digests, and results in a truly random library. Sized, end-repaired genomic
DNA can be ligated to the pWEB Vector directly in the gel. Following
treatment with GELase Preparation, the ligation products can be directly
packaged using MaxPlax Lambda Packaging Extract without further purification.
Using this approach, we have prepared bacterial and eukaryotic libraries
that were ready for screening in 48 hours.
- Sambrook, J. et al. (1989) in: Molecular Cloning: A Laboratory
Manual (2nd ed.), Cold Spring Harbor Laboratory Press, New York.
- Wahl, G.M. et al. (1987) Proc. Natl. Acad. Sci. USA 84,
2160.
- pWEB™ Cosmid Cloning Kit Product Information Sheet,
8/98.
- MutaPlax™ Transgenic Lambda DNA Packaging Kit Product Information
Sheet, 2/96.
- Lai, E. et al. (1991) BioTechniques 11 (2),
212.
- DiLella, A.G. and Woo, S.L.C. (1987) Meth. in Enzymol. 152,
199.
pWEB™ Cosmid Cloning Kit
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