Biomedical Engineering Reference
In-Depth Information
diluted into a series in a secondary set of plates and
screened again. The process is repeated until wells
carrying homogeneous clones corresponding to the
gene of interest have been identified.
There are also several applications where the use
of
degenerate primers
is favourable. A degenerate
primer is a mixture of primers, all of similar sequence
but with variations at one or more positions. This is
analogous to the use of degenerate oligonucleotides
as hybridization probes, and the primers are syn-
thesized in the same way. A common circumstance
requiring the use of degenerate primers is when the
primer sequences have to be deduced from amino
acid sequences (Lee
et al.
1988). Degenerate primers
may also be employed to search for novel members
of a known family of genes (Wilks 1989) or to search
for homologous genes between species (Nunberg
et al.
1989). As with oligonucleotide probes, the
selection of amino acids with low codon degeneracy
is desirable. However, a 128-fold degeneracy in each
primer can be successful in amplifying a single-copy
target from the human genome (Girgis
et al.
1988).
Under such circumstances, the concentration of
any individual primer sequence is very low, so mis-
matching between primer and template must occur
under the annealing conditions chosen. Since mis-
matching of the 3
ally, a random primer method is used for cDNA
synthesis, so there is a greater representation of 5
′
sequences. As discussed above, such libraries are
representative of their source, so certain cDNAs are
abundant and others rare. However, it should be
noted that bacterial expression libraries and many
yeast expression libraries are usually genomic, since
there are few introns in bacteria and some yeasts
and very little intergenic DNA. Efficient expression
libraries can be generated by cloning randomly
sheared genomic DNA or partially digested DNA,
and therefore all genes are represented at the same
frequency (Young
et al
. 1985). A potential problem
with such libraries is that clones corresponding to a
specific gene may carry termination sequences from
the gene lying immediately upstream, which can pre-
vent efficient expression. For this reason, conditions
are imposed so that the size of the fragments for clon-
ing is smaller than that of the target gene, and enough
recombinants are generated for there to be a reason-
able chance that each gene fragment will be cloned
in all six possible reading frames (three in each orient-
ation). Considerations for cloning DNA in
E. coli
ex-
pression vectors are discussed further in Chapter 8.
Immunological screening
-terminal nucleotide of the primer
may prevent efficient extension, degeneracy at this
position is to be avoided.
′
Immunological screening involves the use of anti-
bodies that specifically recognize antigenic deter-
minants on the polypeptide synthesized by a target
clone. This is one of the most versatile expression-
cloning strategies, because it can be applied to any
protein for which an antibody is available. Unlike
the screening strategies discussed below, there is
also no need for that protein to be functional. The
molecular target for recognition is generally an
epitope
, a short sequence of amino acids that folds
into a particular three-dimensional conformation
on the surface of the protein. Epitopes can fold
independently of the rest of the protein and there-
fore often form even when the polypeptide chain
is incomplete or when expressed as a fusion with
another protein. Importantly, many epitopes can
form under denaturing conditions, when the overall
conformation of the protein is abnormal.
The first immunological screening techniques
were developed in the late 1970s, when expression
libraries were generally constructed using plasmid
Screening expression libraries
(expression cloning)
If a DNA library is established using expression
vectors (see Chapter 5), each individual clone can be
expressed to yield a polypeptide. While all libraries
can be screened by hybridization or PCR, as dis-
cussed above, expression libraries are useful because
they allow a range of alternative techniques to be
employed, each of which exploits some structural or
functional property of the gene product. This can be
important in cases where the DNA sequence of the
target clone is completely unknown and there is no
strategy available to design a suitable probe or set of
primers.
For higher eukaryotes, all expression libraries are
cDNA libraries, since these lack introns and the
clones are in most cases of a reasonable size. Gener-
