Gene Cloning

108 Gene Cloning
of your gene is. A common technique is to use a mixture of short random
primers. These will anneal at many points along the first-strand cDNA template
and prime synthesis of the second strand. Random hexanucleotides
or nonanucleotides are often used in this type of application. Another
strategy is to use terminal deoxynucleotidyl transferase to add T residues to
the terminal 3′ hydroxyl end of the cDNA, you can then use a polyA primer
to initiate second-strand synthesis.
Q4.19. How many hexanucleotides would you need to synthesize to have
one of every possible sequence?
4.20 Expressed Sequence Tags
One of the features of a cDNA library is that the DNA fragments that it contains
are representative of the genes which are being transcribed in a particular
tissue at a particular time. This has been exploited as a way of creating
an inventory of expressed genes by partially sequencing large numbers
of the individual clones from a cDNA library. These short, sequenced
regions act as tags identifying individual transcribed regions and are
dubbed expressed sequence tags (ESTs). High-throughput sequencing
methods (Section 7.7) are used to generate raw partial sequences from one
or both ends of individual cDNA clones rapidly and cheaply. They are not
intended as definitive sequence but they are of sufficiently high quality to
identify similar sequences in sequence databases; this will often make it
possible to identify the likely product of the gene they are derived from
using homology searches (Section 8.8). As ESTs represent regions of the
genome which are transcribed into mRNA, they are useful in helping to
identify protein coding regions (Section 8.2) and new genes in DNA
sequencing projects. ESTs have also proved invaluable as genetic markers
in mapping projects (Section 6.6) and, if ESTs have been generated from
both ends of the cDNA clones, they can be used to design PCR primers.
Also, because the ESTs from a given cDNA library represent a snapshot of
the genes expressed in the tissue the library was derived from, they can be
used as a way of comparing gene expression in different tissues at different
times. This basic idea has been developed into the micro-array for global
studies of gene transcription (Section 11.6).
4.21 What are the Disadvantages of a cDNA Library?
cDNA libraries contain only the parts of genes which are found in mature
mRNA. We saw earlier that this can be an advantage because introns will
have been removed. However, if you are interested in the sequences before
and after the gene, for example those which are involved in the regulation
of expression of the gene, you will not find them in a cDNA library. If your
cloning strategy is dependent on expression of a eukaryotic gene, you may