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

302 Edwards and Bartlett
Studies that involve microsatellites usually involve amplification of DNA using
polymerase chain reaction (PCR). The three methods for detection of microsatellites
currently in use are: radioactive PCR visualized by autoradiography; PCR visualized
by silver-stained gels; and fluorescent PCR visualized on an automated DNA fragment
analyzer (7). The current method of choice is fluorescent PCR coupled with analysis
of fluorescent-labeled DNA fragments on polyacrylamide gel electrophoresis in an
automated DNA fragment analyzer. This method considerably increases the throughput
of samples because it uses detection of laser-activated fluorescent products during
electrophoresis and data are then immediately available for analysis. A large number
of microsatellites may be studied at once because as many as 20 PCR products may
be run down one lane of the gel at any one time. This method is also of use if there is a
limited amount of DNA template, for example, small biopsy samples, because it enables
the detection of very low concentrations of PCR products. The fluorescence detection
system has been proven to be at least 10 times more sensitive than autoradiography
and silver staining because of the use of an internal standard the fluorescent system
can more accurately size PCR products and it minimizes stutter bands. This method
also enables detection of LOH even if complete allele loss is not apparent because of
contamination from other cell types. There are also less associated hazards when using
this technique because no handling of radioactivity is required.
LOH and MIS studies commonly are used in cancer research; therefore, tumor and
normal tissue must be separated before DNA can be extracted. Microdissection of
histologically characterized cells from fresh-frozen or paraffin-embedded tissue sections
has become an important technique for the analysis of genetic alterations occurring
in tumors, allowing the separation of normal and tumor cells. Microdissection is a
technique where by tissue sections approx 5-µm thick are stained in toluidine blue and
viewed using a light microscope, and areas of normal/tumor cells are dissected using a
micromanipulator. DNA from the different cells is extracted from the dissected tissue.
The most abundant source of tumor tissue with clinical information is formalin-fixed
paraffin-embedded tissue. Hospitals routinely archive tumors in this fashion, and
although this is acceptable for most pathological processes, it does cause nicks to
form in DNA, this damage to the DNA affects the ability to use it as a PCR template.
The yield of DNA extracted from formalin fixed paraffin embedded tissue is also
significantly diminished, (by about 70% compared to the yield from frozen tissue) (8).
To overcome the problems associated with using archival DNA as a PCR template, the
PCR product should be kept to a minimum size when designing primers, preferably
under 300 bp. To overcome the concentration problem, fluorescent PCR with 40 or
more amplification cycles should be used. Hot start Taq polymerase should also be used
to decrease nonspecific binding of primers to the DNA template and the formation
of primer dimers. With hot start Taq, the amplification reaction only starts after the
initial denaturation step. Therefore, nonspecific PCR products, primer dimer formation,
and background are minimized, maximizing the yield of specific PCR product from
limited template.
The following method is an example of how MIS and LOH can be determined for a
panel of nine microsatellites spanning chromosome 9 using only 9 µL of DNA (approx
15 µg/µL) microdissected from formalin-fixed paraffin-embedded archival transitional
cell carcinomas of the urinary bladder tissue.