Biology Reference
In-Depth Information
ESTs to identify close to full-length cDNA clones of the transcript. These
will usually be in the region of one to four kilobases in length, as opposed
to most ESTs, which are generally 300 to 400 bp in length. The technique
of rapid amplification of cDNA ends (RACE) may then be applied to gen-
erate the missing portions of the cDNA. This technique takes advantage of
the polymerase chain reaction (PCR) to amplify the missing sequence using
mRNA from a specific tissue in which the gene of interest is expressed
(Frohman et al. 1988).
6.2.2 Exon Amplification/Trapping
Not all genes are represented in sequence databases. Thus, alternate
approaches may be necessary to identify candidates in the region of inter-
est. Exon amplification/trapping relies on the fact that the coding regions
of most eukaryotic genes, exons, are separated by noncoding intervening
sequences, introns. The production of mature mRNA involves the removal
of the introns by the process of mRNA splicing to produce continuous
coding sequence for protein translation. The exon/intron boundaries
contain 5¢ donor and 3¢ acceptor splice site consensus sequences. Exon trap-
ping utilizes these sequences to identify clones containing exons. Expres-
sion cloning vectors containing the DNA sequence are used to transfect
a suitably modified eukaryotic cell line, resulting in transcription of the
inserted target DNA into RNA, which then undergoes splicing. The pro-
duction of an abnormal-sized splice product after insertion of a target DNA
fragment indicates the presence of an exon in the cloned DNA fragment
(Duyk et al. 1990).
A major advantage of exon trapping is that it is not dependent on the
expression of the gene. Exons from genes expressed in specific tissues, or
at particular stages of development, are isolated with the same efficiency as
genes that are more widely expressed (Church et al. 1994). Disadvantages
of exon trapping are that genes with single exons and exons in the 3¢ and
5¢ portions of the gene will be missed. Specialized vectors have been devised
to help overcome this problem (Krizman et al. 1995).
6.2.3 Phenotype Rescue
More recently, “phenotype rescue” in the mouse has helped to rapidly
narrow the interval within which a gene of interest is located. In this pro-
cedure, BAC clones from the contig to which the gene has been mapped
are injected into fertilized mouse oocytes that are homozygous for a muta-
tion in this gene (e.g., a gene causing hearing impairment). Identification of
a transgenic mouse with normal hearing (i.e., an offspring for which the
phenotype reverts to normal or is “rescued”), suggests that the injected
BAC clone contains the gene. This approach was used to identify
Myo15
as
the defective gene in the
shaker-2
mouse (Probst et al. 1998). Almost imme-
diately, mutations in the human homologue,
MYO15
, were shown to be
