John M. S. Bartlett.pdf - Bio-Nica.info

Mutation and Polymorphism Detection 291
DNA may be extracted from fresh, frozen, or archival material. High-quality DNA
should be easily retrieved from fresh or frozen material, for example, tissue or blood.
Extraction of good-quality DNA from archival material is more difficult; however, the
most abundant source of clinically available tissue is archival formalin-fixed paraffinembedded
tissue. This process not only drastically decreases the yield of DNA in
comparison with fresh or frozen material but also damages the DNA, often making it
unsuitable as a DNA template. Chapter 43 discusses how archival DNA can be used for
microsatellite analysis by keeping the PCR product size small.
In summary, for successful mutational analysis a significant quantity of high-quality,
pure DNA is required. Low quantities of DNA make analysis difficult, and poor-quality
or contaminated DNA may cause false results.
3.2. PCR Artifacts
Although PCR is the recognized method for increasing the quantity of DNA required
for polymorphism or mutational analysis, PCR can in itself introduce problems.
Contamination by foreign DNA and cross-contamination is often introduced at this
stage of the process. With appropriate care, that is, gloves, aseptic technique, and
clean pipette tips between every sample, this should be limited. However, this is
a particular problem with DOP-PCR. DOP-PCR is very sensitive to contamination
because degenerate primers in the reaction will amplify DNA from any source present
in the tube. However, the presence of DNA in the blank control after DOP-PCR does
not always indicate contamination, and the low temperature in the DOP-PCR can result
in DOP primers attaching to each other and replicating (15). If this occurs, then a
second PCR amplifying only the region of interest should not be possible. It is therefore
recommended that blanks should be tested by sequential PCR after DOP-PCR before
samples are discarded.
Other problems created by PCR are the addition of an extra base (normally adenine) at
the end of the DNA sequence and the creation of shadow bands (discussed in Chapter 44).
In summary, although PCR solves the problem of insufficient DNA for analysis, with
its use comes an assortment of additional problems.
4.1. Applications
Mutational analysis has many applications, for example, markers of disease, cancer
research, and genotyping. Mutations are responsible for diseases, such as sickle cell
anemia (1), cystic fibrosis (1), and adrenal leukodystrophy (1). An expansion of the
CAG repeat in exon 1 of the androgen receptor alone can result in Kennedy’s disease,
spinocerebellar ataxia, or fragile X syndrome (15). Therefore, polymorphism and
mutational analysis are of use in diagnosing these diseases.
Detection of polymorphism and mutational analysis is also widely used in the field
of cancer research. Mutation detection has demonstrated that p53 function is lost in
approximately 50% of all cancers and that the loss of function is caused by point
mutations (16). It also demonstrates that a mutation in BRCA 1 or BRCA 2 increases
susceptibility to breast and ovarian cancer (17). Microsatellite analysis is used in cancer
research to study LOH and microsatellite instability. LOH studies have demonstrated
most genetic alterations that occur in bladder cancer are on chromosome 9 (18) and that
microsatellite instability is commonly found in colorectal cancer (13).