WORKING WITH CLONES
Context
In genomes without a determined genomic sequence, cDNAs may
be cloned by screening with oligos designed from a gene sequence
in a homologous organism, or by screening with antibodies raised
to an isolated protein. Then isolated cDNA can be used to
screen genomic libraries by hybridization. The cDNA cloning
step may be replaced by EST Database searching if that level of
sequence characterization is available for the organism. If
there is genomic sequence available, then any section of DNA
relative to the cDNA sequence (e.g. the transcriptional
promoter) may be directly targeted by PCR. Even in the absence
of genomic sequence, the genomic DNA immediately flanking the
cDNA might be retrieved by inverted or anchored PCR approaches.
Hence, the number of scientists needing to screen libraries or
work with large insert cloning vectors has been radically
reduced. This document contains a summary of large insert
cloning vectors and procedures that we no longer teach due to
the relative infrequency of its their use.
An example of a case where one might still work with large
insert clones is to conduct transgenic experiments where very
large segments of the genome are introduced. In these cases,
one of the relevant genome centers may maintain a large insert
library from which you may directly order the relevant clone.
Lambda clones.

Lambda gt10 (used for cDNAs)
Lambda gt10 is a vector that can carry cDNA inserts of
between 0 and 5kb. The cDNA is joined to an EcoRI site in the
vector through the use of EcoRI linkers. Exclusion of
uninserted clones is achieved by growth on a special host strain
(Hfl-) that causes the phage to always lysogenize if it is cI+.
Therefore, if uninserted lambda gt10 infects its Hfl- host, it
does not make a plaque, and disappears for all practical
purposes. However, insertion in the EcoRI site disrupts the cI
gene, precluding lysogeny. Therefore, only the phage carrying
inserts make plaques.
Lambda gt10 clones do not have to be
propagated on Hfla- hosts. DNA preparation requires a lytic
growth (see below). For virtually all purposes, the insert will
have to be removed and subcloned into something else.
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Lambda gt11
Lambda gt11 is a similar vector designed for use with
antibody screening.
Ref: Young and Davis, PNAS 80,1194.
The object was to carry cDNAs so that they are expressed as
a lac Z fusion under lac control in a lambda lysogen. Induction
with IPTG will cause expression of the fused protein in the
lysogenic colonies which can then be screened with antibodies.
Use of the lac Z fusion provides the following benefits
over using just a transcriptional fusion to the lac promoter.
1. Peptide fragments fused to lac Z are less susceptible to
degradation by bacterial enzymes.
2. The fused protein is extremely large and therefore easy to
identify on gels.
3. One gets consistent translational efficiency.
4. Inactivation of betagalactosidase activity gives a color test
for the presence of insertions.
The vector can hold up to a 8.3 Kb insert.
When one prepares inserts for lambda gt11, generally the
insert is fragmented at random to assure joining to
betagalactosidase in all possible reading frames. Note that
there is no expectation to produce a functional protein, just
antigen. For most further purposes, the insert will be lifted
out of the vector, sequenced, and used as a basis to hunt for a
full length cDNA.
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The genetic interactions involved in the system include the
following genes:
Phage:
cI857
S100
lac Z+
-
temperature sensitive lambda repressor
amber mutation in lysis gene
Host for lytic growth (for counting plaques and conducting a
blue/white color test for insertion):
lac Zlac I+
hsd R- hsd M+
supF
K restriction system
cancels effect of S100
Host for lysogeny (for the actual antibody screening or for DNA
preparation):
lac ZhflA -
high frequency lysogeny
Growth of lambda DNA
A lysogen is the preferred form for preparing lambda DNA if
you have the temperature sensitive cI gene and the S100
mutation. You grow the cells up and then heat to get a
synchronous transition to lytic growth. The S100 mutation keeps
the cells from lysing, so phage production goes on much longer
than it normally would. The cells can then be harvested and
broken open to yield a high titer of phage.
If the lysogenic system is unavailable, then you have to do
a lytic growth. During a lytic growth lambda is grown by adding
a small innoculum of phage to a larger innoculum of bacteria.
As the culture grows, the bacteria double every generation,
whereas the phage increase by 50-100x every generation.
Consequently, at some point the phage overtake the bacteria and
lyse the entire culture.
If the phage overtake the bacteria too early, there won't
be many bacteria to grow in, so the yield will be low. If the
bacteria grow too long before becoming infected, they become
refractory to infection, and the culture never lyses. So the
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exact ratio of phage to bacteria at the beginning is critical;
and, unfortunately, the optimal ratio for each clone is not the
same. Furthermore, if you are not there to observe the lysis of
the culture and harvest the phage immediately, the phage will
inactivate themselves trying to infect the cell debris, and the
yield will be low. Consequently lytic growth of lambda clones
tends to be a frustrating process. I recommend starting 4
different flasks at different phage:bacteria ratios and just
harvesting the ones that appear to lyse on schedule. There are
also plate lysate methods of making lambda DNA that similarly
have timing problems.
lambda ZAP
Lambda ZAP is a commercial vector (Strategene) that
provides another alternative for DNA preparation. The lambda
vector contains a complete copy of a phagemid vector.
(Phagemids are vectors that can be grown as either an M13 phage
or as a plasmid). The multiple cloning region is inside of the
phagemid. Coinfection of the lambda Zap containing cell with an
M13 helper phage causes the phagemid to be excised from the
lambda DNA and repackaged in an M13 phage head. These phage can
be subsequently used to reinfect cells and establish the clone
as a plasmid.
Some vectors for genomic cloning
Lambda EMBL3
Lambda can hold up to 20Kb of foreign material. In this
case the uninserted vector is too small to successfully package
into a lambda phage head, so the vector is prepared with a
"stuffer" fragment to fill out the DNA to an acceptable size.
Cloning consists of replacing the stuffer with insert and then
packaging as before. A particularly popular lambda cloning
vector is lambda EMBL3. Its stuffer fragment can be genetically
excluded from recloning, thus avoiding having to physically
purify the lambda arms from the stuffer before ligation to the
insert.
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Exclusion of the stuffer is by spi- selection. A special
host is used (lysogenic for the unrelated phage P2) that
precludes plaque formation by lambda carrying its normal
recombination functions, red+, gam+. Such phage are said to be
spi+ (sensitive to P2 interference). EMBL3 is spi+, and cannot
form plaques on this host. The red and gam genes of EMBL3 are
carried in the stuffer fragment, so replacement of the stuffer
with insert causes a conversion to spi-, which will form plaques
on this host. Commercial libraries may be supplied with more
than one host strain. The construction strain if supplied is
irrelevant since you're getting a library that has already been
passed through that strain and amplified.
EMBL3 can accept inserts of between 12 and 20 Kb. There is
no restriction enzyme that will cleave genomic DNA so that all
sequences fall on fragments of this size. Note that any
sequence that falls on a fragment outside of the acceptable size
range will never appear in the library (except as a multiple
insert).
To prepare the insert, the genomic DNA is subjected to
partial restriction digestion with a frequently cutting enzyme,
usually Sau3A. Several different reactions are carried out to
different extents of digestion. Preparative gel electrophoresis
is then used to purify the fraction from all digests that falls
between 15 and 20Kb. This DNA is then cloned by ligation to the
compatible BamHI sites on the EMBL3 arms. The idea is to
exclude multiple inserts because they would make the DNA too big
to package. However, this is variably successful, and if
someone else made the library you have no idea how carefully
they did the size cut. So individual clones will always have to
be characterized for multiple inserts through a Southern blot
experiment.
Another feature of EMBL3 is the presence of SalI sites just
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outside of the linker on either side. Since SalI is an
infrequent cutter in mammalian DNA, this often means that the
insert can be removed as a single Sal fragment.
Complexity is the number of different clones in a library,
not the number of total clones. When the in vitro packaged
material is originally plated, every plaque should represent a
different clone, and the number of plaques is a measure of the
complexity. However, most libraries are then "amplified" before
screening. In this process, the phage from the original set of
plaques are harvested and all mixed together. This increases
the total number of phage, but not the complexity. In fact, the
complexity may drop, because some clones may not have replicated
as well as others. If the library is repeatedly amplified, a
very small number of aggressive clones may outgrow all of the
others. The advantage of amplification is that aliquots of the
library can now be plated and screened many different times.
All commercial libraries are extensively amplified. In
practice, you can't really be guaranteed of finding any
particular clone in any particular library.
Complexity of clones required to screen = ln(1-P)/ln[1-(I/G)]
Where P is the probability of finding what you want,
and I/G is the ratio of insert size to genome size.
Supposing that you have the clone you want, you still have
a task of recovering from the 20 kb of insert the smaller piece
of DNA of interest. The traditional way is to restriction map
the insert and probe to identify the fragment of interest. Then
one would cut that fragment out of a gel and subclone it into a
plasmid in preparation for sequencing. Now it is possible to
directly sequence DNA within a DNA the size of lambda, so you
could try to directly sequence from a primer in the known region
and then lift the segment of interest out by PCR.
YACs
YACs are Yeast Artificial Chromosomes. These clones can
carry inserts as large as 1 Mb. They are screened by PCR by a
commercial entity. The clones can only be grown in yeast. The
yield of DNA is low, and they have to be separated from the
yeast chromosomes by pulsed field gel electrophoresis. YACs are
intermediates in the characterization of large genomes (complete
human and mouse libraries are available). A gene might be known
to be on a particular YAC by positional cloning information. I
one received a YAC, the first order of business would be to get
the region of interest out of the YAC and into some vector that
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is more tractable.
Cosmids
Plasmids are capable of carrying very large inserts,
however it is difficult to get efficient transformation with
large plasmids. Cosmids are a solution to this problem in which
the lambda in vitro packaging system is used to introduce
molecules into E. coli that will subsequently grow as ordinary
plasmids.
Lambda contains a double stranded molecule in the phage
head containing 12 base cohesive ends. Upon introduction into
the host, the ends are ligated resulting in a double stranded
circular molecule. This molecule replicates, directs expression
of the phage genes, and then switches to a mode of replication
that generates a long concatemer containing a tandem array of
lambda genomes. During packaging, the junctions between the
lambda genomes (called cos sites) are recognized by an enzyme
called ter, and cleaved to remake the linear lambda genomes with
cohesive ends.
A cosmid is simply a plasmid containing the lambda cos
site. If the plasmid is linearized and ligated into a
concatemer that places the cos sites about 50 Kb apart, then in
vitro packaging can be conducted to pack lambda length molecules
into lambda phage heads. These molecules will have lambda
cohesive ends and upon introduction into E. coli will
circularize just as lambda does. However, at this point there
will be no lambda genes to express, so there will be no further
continuation of the lambda life cycle. Instead, the plasmid ori
will take over and propagate the circle as an ordinary plasmid.
The cosmid contains a drug resistance marker to facilitate
selection for cells containing the cosmid.
Cosmids have to be prepared by a method that inhibits both
multiple insertions and inclusion of multiple copies of the
cosmid vector. Multiple inserts are prevented by phosphatasing
the sized insert material. The strategy shown below excludes
concatemerization of vector with ligation conditions that allow
ligations of cohesive (BamHI + Sau3A) ends without ligation of
blunt (SmaI) ends.
Ref: Molecular Cloning by Sambrook, Fritsch, and Maniatis
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P1
P1 is another bacteriophage that is being adapted to
cloning large inserts. P1 DNA also contains a packaging site
(called pac) which like lambda cos is recognized during
packaging and cut to make one end of the packaged DNA. However,
unlike lambda, the P1 packaging system simply packages a
headfull (110-115 Kb) before making its second cut without
respect to the sequence. Consequently, one can ligate any DNA
next to a pac site and get it packaged into a P1 phage head. The
pac site is provided by the left arm in the diagram below.
To facilitate maintenance of the DNA after it is injected
by the P1 phage into E. coli, a right arm is also provided
carrying an origin of replication and an antibiotic resistance
gene. Circularization is provided by the presence of a loxP
site in each arm. A special host strain expressing the P1 cre
recombinase is used to recombine the two loxP sites and make a
circle which then is propagated as a plasmid.
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The
stuffer in the right arm is to provide some leeway in the size
of the insert. Without the stuffer, only inserts in a +- 5Kb
size range would be large enough to package, and small enough to
have two loxP sites in the packaged unit. Exclusion of clones
containing multiple vector fragments is achieved by
phosphatasing both ends of each vector arm. Exclusion of
multiple inserts is because two inserts will put the second loxP
site too far away to get packaged on the same fragment with the
first loxP site.
Another feature of the pictured vector is the inclusion of
an inducible high copy ori that can be used to replicate the
clone up to a larger mass to support DNA preparation.
Finally, the insert site interrupts a sacB gene(not shown) which
kills the host when grown on sucrose. Therefore, growth on
sucrose kills all clones without inserts, thus providing a
direct selection for inserts.
BACs
Ref: Shizuya et al., Cloning and stable maintenance of 300kilobase-pair fragments of human DNA in Escherichia coli using
an F-factor-based vector.
BAC stands for "Bacterial Artificial Chromosome".
The vector (essentially a plasmid) uses an F factor ori because
this produces a copy number of one. Large inserts are more
stable at low copy number.
The selectable marker is for chloramphenicol resistance.
Large inserts are directly ligated into the cut vector and the
ligation mix is electroporated into E. coli. The host strain is
especially competent for electroporation by large DNA, and is
deficient in hsdMRS, mcrABC, mrr, recBC, sbcBC and recJ or recA.
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Because the DNA is isolated as a supercoil, it is less shear
sensitive than a YAC.
The vector has multiple infrequent cutting enzyme sites
surrounding the cloning sites (HindIII or BamHI) as well as
flanking T7 and Sp6 promoters for making riboprobes.
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Screening libraries by PCR.
Screening a library banked as individual clones:
Screening a library banked as individual clones:
For final location of the desired YAC clone, pools are
screened representing specific rows and columns on a plate.
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Screening a bulk library:
Bulk lambda library of complexity 3 x 106
--> 30 tubes of titer 105 pfu
Amplify each tube and screen by PCR
From positive tube make 48 tubes of titer 104 pfu.
Amplify and screen by PCR.
etc.
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