Biomedical Engineering Reference
In-Depth Information
T3
T7
A
J
red
+
gam
+
14 kb
N
OPQSR
λ
DASH
(a)
20 kb
9 kb
T7
T3
Fig. 6.4
The replacement vectors
λDASH and λFIX. Promoters
specific for the bacteriophage T3
and T7 RNA polymerases are
located adjacent to the cloning
sites, allowing RNA probes to be
generated that correspond to
each end of the insert.
A
J
red
+
gam
+
14 kb
N
OPQSR
λ
FIX
20 kb
9 kb
(b)
these vectors are maintained as high-copy-number
plasmids in
Escherichia coli
, they have a tendency
to be unstable, undergoing deletions that favour
increased replication. Most workers also find that
plaques give less background hybridization than
colonies, so screening libraries of phage-
equally well, e.g. variations in target DNA size or
sequence may affect replication of a recombinant
phage, plasmid or cosmid. Therefore, when a library
is put through an amplification step, particular
recombinants may be increased in frequency,
decreased in frequency or lost altogether. The devel-
opment of modern vectors and cloning strategies
has simplified library construction to the point
where many workers now prefer to create a new
library for each screening, rather than risk using a
previously amplified one. Furthermore, pre-made
libraries are available from many commercial
sources and the same companies often offer custom
library services. These libraries are often of high
quality and such services are becoming increasingly
popular.
The highest-capacity vectors - BACs, PACs and
YACs - would seem to be ideal for library con-
struction because of the very large insert sizes.
However, such libraries are generally more difficult
to prepare, and the larger inserts can be less than
straightforward to work with. Unless the genomic
target sequence is known to be very large and needs
to be isolated as a single clone,
recombin-
ants by plaque hybridization gives cleaner results
than screening cosmid libraries by colony hybridiza-
tion (see p. 104). It may also be desired to retain and
store an
amplified genomic library
. With phage, the
initial recombinant DNA population is packaged
and plated out, and can be screened at this stage.
Alternatively, the plates containing the recombin-
ant plaques can be washed to give an amplified library
of recombinant phage. The amplified library can
then be stored almost indefinitely, because phage
have a long shelf-life. The amplification is so great
that samples of the amplified library can be plated
out and screened with different probes on hundreds
of occasions. With bacterial colonies containing
cosmids, it is also possible to store an amplified
library, but bacterial populations cannot be stored
as readily as phage populations, and there is often
an unacceptable loss of viability (Hanahan &
Meselson 1980). A word of caution is necessary,
however, when considering the use of any amplified
library. This is due to the possibility of
distortion
.
Not all recombinants in a population will propagate
λ
replacement vectors
or cosmids remain the most appropriate choice for
many experiments. The main application of BAC,
PAC and YAC libraries is for genome mapping,
sequencing and the assembly of clone contigs.
λ
