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elements usually found in arthropod genes. Thus, a cDNA clone will contain the
DNA sequence of the protein of interest, but it will lack introns and probably
not contain the control sequences that regulate gene expression. cDNA cloning
is used to produce a cDNA library or to produce probes for screening genomic
libraries.
A cDNA library allows the genetic engineer to clone only those genes that are
active at a specific time or in specific tissues . Genes that are not actively tran-
scribed into mRNA will not be represented in the cDNA library. Thus, a cDNA
library usually contains fewer clones than a genomic library. The gene of interest
may occur in a frequency of one in 10 3 or one in 10 4 clones. By contrast, a single-
copy gene may be present in a genomic library in a frequency of only one in 10 5
to one in 10 6 clones. Another benefit of a cDNA library is that it is possible, if an
appropriate expression vector is used, to express a gene in a host such as E. coli
or yeast. This enables the genetic engineer to produce large amounts of a spe-
cific gene product.
The quality of a cDNA library depends on the quality of the mRNA used as
the template and the fidelity with which it can be reverse transcribed into
cDNA. Messenger RNA, together with a suitable primer, and a supply of deoxy-
ribonucleoside triphosphates can be converted into a ss DNA molecule with the
enzyme reverse transcriptase ( Figure 6.9 ).
The cDNA cloning process involves two steps: 1) the first strand of cDNA is
produced by reverse transcription, and 2) a strand that is complementary to the
first strand is then synthesized using DNA polymerase so that a ds cDNA mol-
ecule is produced. The primer used to synthesize the first DNA strand is usually
an oligonucleotide consisting of deoxythimidine (dT) residues because it can
hybridize to the 3 poly(A) tails of template mRNA and potentially give rise to
full-length copies of the complementary DNA strand. Once the ds cDNA mole-
cule has been synthesized, it can then be inserted into a plasmid or phage vector
that is capable of replicating in E. coli and, in some cases, of being translated
into a protein.
A key to producing cDNA is the enzyme reverse transcriptase . Reverse tran-
scriptase is capable of two functions in vitro : a polymerase activity and a ribo-
nuclease H activity. The polymerase activity requires 1) a template RNA molecule
hybridized to a DNA primer with a 3 -OH group and 2) all four dNTPs to synthe-
size a DNA molecule that is a faithful complement of the mRNA.
Cloning a cDNA library is more complex than cloning a genomic library into
λ or cosmids. Before beginning the process, the goals of the project must be
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