Biology Reference
In-Depth Information
that contain polymorphic markers known to map to the interval in the
region of interest. Now, because of the progress of the Human Genome
Project, YAC contigs of most regions of the genome are available online.
Identification of unique DNA sequences in the YAC clones allows their
order to be determined leading to a physically contiguous series of over-
lapping YAC clones (contig).
A concern with YACs is that the DNA inserts are often chimeric, i.e.,
contain DNA fragments from noncontiguous regions of the genome. In
addition, YAC clones are often unstable, undergoing rearrangements in
propagation. For this reason, once the region containing a gene of interest
has been narrowed down sufficiently, bacterial artificial chromosomes
(BACs) and/or bacteriophage P1 artificial chromosomes (PACs), which
replicate stably, are used to establish a contig of the interval of interest.
Identification of a gene in a contig may occur quite rapidly if the region
of interest has previously been screened in a different study. For example,
the
DFNB3
locus mapped to chromosome 17p11.2, a region that was known
to contain the gene for hereditary motor sensory neuropathy type 1a
(
CMT1A
) (Murakami and Lupski 1996) and was also the location of the
microdeletions responsible for Smith-Magenis syndrome (Juyal et al. 1996).
This allowed screening of genes that had previously been found in that
region (Murakami et al. 1997), one of which was the homologue of the gene
responsible for the mouse deafness mutant
shaker-2
(Liang et al. 1998).
6.2 Transcript Mapping
Once a physical contig is established for a particular region known to
contain a gene of interest, the next step in the mapping process is the
identification of expressed gene sequences or transcripts within that region
(Giersch and Morton, Chapter 3).
6.2.1 Expressed Sequence Tags (ESTs)
Partial sequences of genes identified from cDNAs are known as expressed
sequence tags (ESTs) (Adams et al. 1991). More than 50,000 human ESTs
have been identified, many of which have been mapped and deposited in
the DNA sequence database, dbEST (Boguski 1993; Boguski and Schuler
1995; Schuler et al. 1996). Recently 30,000 of these ESTs have been assem-
bled in an integrated map (Deloukas et al. 1998), nearly twice as many as
three years earlier (Berry et al. 1995). This resource contains most of the
genes that encode proteins of known function. By selecting the chromoso-
mal region of interest in the database, ESTs mapping to that region can be
identified.
ESTs, however, represent only partial sequences of genes and it is nec-
essary to identify the full sequence to be able to reliably screen for muta-
tions in a specific gene. This is done by screening a cDNA library with the
