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Mutation and Polymorphism Detection 289
development and if this technique is successful should enable the investigation of
thousands of SNPs in parallel (8). However, currently there are still limitations
associated with it. A recent mapping study analyzed 500 SNPs using array technology
and only 70% of sites analyzed were genotyped correctly (9). This limitation has been
broached by using enzymatic reactions with the oligonucleotide arrays to enhance
specificity of hybridization, for example, array primer extensions are being developed
that use the principle of allele specific single-base primer extension. It has been
demonstrated that this method is more specific at identifying unknown mutations
in a known sequence (deletions, transversions, and up to two-base insertions) that
can be readily found, using arrayed primer extension reactions (10). However,
promising mutational analysis using oligonucleotide microarrays still remains in the
developmental stage.
2.2. Detection of Large Inserts or Deletions
Large inserts or deletions may be detected by the methods discussed above, such as
SSCP, enzyme cleavage systems, or amplification refractory mutation system discussed
in detail in Chapter 47. However, currently the most commonly used detection system
is either radioactive or fluorescent-based automated sequencing. Although many of
the above mentioned available screening methods detect in the region of 90 to 98%
of mutations (whether SNPs, deletions, or insertions) there are still mutations that are
missed. Therefore, currently the only way to assure that every mutation is found is to
sequence the region of interest, although this method is both costly and time consuming
and requires high-quality pure DNA.
2.3. Detection of Loss of Heterozygosity (LOH)
and Replication Error Phenotype
Microsatellite loci have a high degree of polymorphism that is caused by problems
associated with copying repetitive sequences of DNA. Microsatellites are popular
genetic markers because of their abundance and high level of allelic variation, and
expansion of microsatellite trinucleotide repeats have been demonstrated to cause
several human genetic disease (11). Microsatellite analysis is also useful for detection
of LOH and replication errors in tumors (12,13). LOH is detected as a reduced intensity
or total loss of one or more bands in the tumor DNA compared with normal DNA
form the same individual (12). Replication errors are detected as changes in the length
of the microsatellite sequences in the tumor DNA compared with normal DNA (13).
Mutation polymorphism detection of microsatellites basically involves amplifying
the microsatellite loci and estimating the size of the PCR product. The most common
techniques used to estimate the size of the microsatellite product involves separation
of products by gel electrophoresis. The most common method used to visualize PCR
products on an agarose gel is ethidium bromide staining. Staining with ethidium
bromide is not a routine method used for microsatellite analysis because it has low
sensitivity and does not provide a permanent record. Microsatellite analysis is more
commonly performed using polyacrylamide gels because they give better separation
than agarose gels. Visualization of DNA on a polyacrylamide gel may be achieved
by silver staining, radioactive-labeled DNA visualized by autoradiography, or laseractivated
fluorescent-labeled DNA detected by an automated sequencer. Silver staining
can be difficult to control, and use of radiation within the laboratory setting is becom-