Biomedical Engineering Reference
In-Depth Information
for gene isolation. However, as well as being useful
for the isolation of specific fragments, the PCR can be
used to generate libraries, i.e. by amplifying a repres-
entative collection of random genomic fragments.
This can be achieved using either random primers
followed by size selection for suitable PCR products
or a strategy in which genomic DNA is digested with
restriction enzymes and then linkers are ligated to
the ends of the DNA fragments, providing annealing
sites for one specific type of primer (e.g. Zhang
et al
.
1992, Cheung & Nelson 1996). These techniques
are powerful because they allow genomic fragment
libraries to be prepared from material that could not
yield DNA of suitable quality or quantity for conven-
tional library construction, but competition among
the templates generally does not allow the produc-
tion of a truly representative library.
splicing does not occur in bacteria, eukaryotic cDNA
clones find application where bacterial expression of
the foreign DNA is necessary, either as a prerequisite
for detecting the clone (see p. 109) or because ex-
pression of the polypeptide product is the primary
objective. Also, where the sequence of the genomic
DNA is available, the position of intron/exon bound-
aries can be assigned by comparison with the cDNA
sequence.
cDNA libraries
Under some circumstances, it may be possible to pre-
pare cDNA directly from a purified mRNA species.
Much more commonly, a
cDNA library
is prepared
by reverse-transcribing a population of mRNAs and
then screened for particular clones. An important
concept is that the cDNA library is representative of
the RNA population from which it was derived.
Thus, whereas genomic libraries are essentially the
same, regardless of the cell type or developmental
stage from which the DNA was isolated, the con-
tents of cDNA libraries will vary widely according
to these parameters. A given cDNA library will also
be enriched for abundant mRNAs but may contain
only a few clones representing rare mRNAs. Fur-
thermore, where a gene is differentially spliced, a
cDNA library will contain different clones represent-
ing alternative splice variants.
Table 6.2 shows the abundances of different classes
of mRNAs in two representative tissues. Generally,
mRNAs can be described as abundant, moderately
abundant or rare. Notice that, in the chicken
oviduct, one mRNA type is superabundant. This
encodes ovalbumin, the major egg-white protein.
Therefore, the starting population is naturally so
enriched in ovalbumin mRNA that isolating the
ovalbumin cDNA can be achieved without the use of
a library. An appropriate strategy for obtaining such
abundant cDNAs is to clone them directly in an M13
vector, such as M13 mp8. A set of clones can then be
sequenced immediately and identified on the basis
of the polypeptide that each encodes. A successful
demonstration of this strategy was reported by
Putney
et al.
(1983), who determined DNA sequences
of 178 randomly chosen muscle cDNA clones. Based
on the amino acid sequences available for 19
abundant muscle-specific proteins, they were able to
cDNA cloning
Properties of cDNA
cDNA is prepared by reverse-transcribing cellular
RNA. Cloned eukaryotic cDNAs have their own
special uses, which derive from the fact that they
lack introns and other non-coding sequences pre-
sent in the corresponding genomic DNA. Introns
are rare in bacteria but occur in most genes of
higher eukaryotes. They can be situated within the
coding sequence itself, where they then interrupt
the collinear relationship of the gene and its encoded
polypeptide, or they may occur in the 5
untrans-
lated regions. In any event, they are copied by RNA
polymerase when the gene is transcribed. The
primary transcript goes through a series of process-
ing events in the nucleus before appearing in the
cytoplasm as mature mRNA. These events include
the removal of intron sequences by a process called
splicing.
In mammals, some genes contain numer-
ous large introns that represent the vast majority of
the sequence. For example, the human dystrophin
gene contains 79 introns, representing over 99% of
the sequence. The gene is nearly 2.5 Mb in length
and yet the corresponding cDNA is only just over 11
kb. Thus, one advantage of cDNA cloning is that in
many cases the size of the cDNA clone is significantly
lower than that of the corresponding genomic clone.
Since removal of eukaryotic intron transcripts by
′
or 3
′
