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

290 Edwards and Bartlett
ing less popular in current years because of the associated health risks. Therefore,
fluorescent-based methods are currently the method of choice, that is, labeling of DNA
using fluorescent-labeled primers or nucleotides. Fluorescent technology eliminates the
handling of radioactivity, makes it easier to interpret stutter bands and laser-activated
detection of fluorescent products during electrophoresis, allows immediate detection of
signal, permits rapid data analysis, and provides permanent records of results. The use
of fluorescent technology has allowed development of techniques that greatly increase
the speed by which microsatellite analysis can be performed by increasing throughput.
For example, a rapid way to screen for microsatellites is described in Chapter 42. This
chapter discusses multiplex touchdown PCR, which allows amplification of multiple
microsatellite loci simultaneously. Use of different fluorescent-labeled primers then
allows identification of each loci. Similarly, Chapter 43 describes the use of fluorescent
technology to increase throughput of LOH analysis by labeling PCR products with
differently labeled fluorescent primers.
3. Technical Constraints
All of the above methods require good-quality DNA; therefore, in most cases before
mutational analysis can begin RNA or DNA must be extracted from the sample and a PCR
performed to provide DNA of sufficient purity and quantity for mutational analysis.
3.1. Problems Associated with Quality and Quantity
of Extracted DNA or RNA
If the amount of available DNA or RNA is limited and therefore insufficient for
use in mutational analysis, PCR or RT-PCR may be used to increase the quantity of
DNA. If multiple regions of interest are to be investigated, then it may be necessary to
globally amplify DNA by degenerate oligonucleotide-primed PCR (DOP-PCR) (14).
The use of this technique in mutational analysis is described in Chapter 45.
The purity of extracted DNA or RNA for subsequent mutational analysis is extremely
important. Contamination with other cellular components, such as protein, or chemicals,
such as ethanol, can easily be removed after the extraction process using various
cleanup protocols. However, contamination with foreign DNA is not dealt with as
easily. Contamination at this stage of the process with either foreign DNA from external
sources or from within the sample will be amplified as the process proceeds and
can result in mutational analysis being conducted on the wrong DNA. Precautions
should be taken to limit cross-contamination and external contamination as with any
PCR. Methods for increasing the purity of RNA and decreasing the introduction of
contamination during RT-PCR are discussed in Chapter 46. Contamination may also be
a problem from within the tissue, for example, if studying cancer genetics, normal cells
may contaminate a tumor cell population. Microdissection is a technique that allows
separation of normal and tumor cells, and Chapter 43 discusses use of this method
in microsatellite analysis.
For mutational analysis, DNA is required to be of high quality because poor-quality
DNA, for example, degraded or nicked, could result in false results. If performing
RT-PCR from RNA, RNA should be extracted from fresh tissue or from tissue snap
frozen on removal to decrease RNA degradation. Although kits now are available for
extraction of RNA from archival material, the quality of RNA retrieved is often poor.