Advances in Understanding Genetic Changes in Cancer Impact on Diagnosis and Treatment Decisions in the 1990s (1992) / Chapter Skim
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2. Testing for Tumor-Specific Genetic Markers
2. Testing for Tumor-Specific Genetic Markers
Pages 8-24
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From page 8... ...
Most importantly, morphologic evaluation is subjective and may be prone to observer variability. Additionally, morphologic evaluation is relatively insensitive, allowing detection of tumor cells only if their frequency is greater than 1-10% in most circumstances.
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From page 9... ...
Many of the methods now used to detect tumor markers identify changes in DNA structure or sequence directly. However, because genetic information encoded in DNA is transcribed into RNA and finally translated into protein, tests that assess RNA sequence or quantity, or protein structure, function, or quantity, are also informative in various situations.
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From page 10... ...
10 - o o sit ._ - o On Em ._ 4 o Cal Cd I a' o 3 ._ 50 .
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From page 12... ...
Other abnormalities seen in tumor cells include double minute chromosomes (dmin) , small fragments of chromosomes that are present in numerous copies and contain amplifications of specific genes.
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From page 13... ...
If the nucleotide sequence of the probe is complementary to a sequence in one of the two strands of the dissociated target DNA, it can bind to that sequence, a process termed hybridization. Techniques have been developed that permit hybridization of specific DNA sequences in metaphase chromosomes or intact nuclei by incubating preparations with tagged probes complementary to the sequences of interest.
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From page 14... ...
For probes directed at individual genes, the probability of this occurring is about 1%; with centromeric probes, which recognize a larger target, the frequency increases to about 3 to 8%. It may be possible to alleviate this problem by using special confocal microscopes capable of "optical" sectioning, thereby allowing resolution of vertically superimposed hybridization targets, but this is currently a relatively slow procedure.
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From page 15... ...
Ploidy Analysis Two other strategies that provide certain kinds of information about the state of chromosomes in tumor cells involve flow cytometry and image analysis. Application of these techniques is based on the correlation of higher or lower DNA content in cancer tissues, due either to aneuploidy or to increased DNA synthesis and cell division, compared to nonmalignant tissues.
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From page 16... ...
Treatment of DNA with a restriction enzyme results in a set of DNA fragments of varying size, which can be separated by electrophoresis on agarose gels. Individual restriction fragments within the complex array of differently sized fragments resolved by the gel can be identified by using a technique called Southern blot hybridization (4)
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From page 17... ...
The technique is relatively slow, requiring about one week from receipt of the specimen under the best of circumstances. Despite these disadvantages, Southern blotting has proved invaluable in recent years for screening tissues for various forms of DNA rearrangements, including translocations, deletions, antigen receptor gene rearrangements, and gene amplifications.
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From page 18... ...
Ultimately, these problems are surmounted only by identification of the affected gene, which allows direct testing for the mutant allele. RFLP analysis has also been used to identify regions of the genome likely to contain tumor suppressor genes.
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From page 19... ...
This technique permits in vitro production of many copies of a defined DNA sequence up to several kilobases long, provided DNA sequences flanking this region on both sides are known. The technique requires the enzyme DNA polymerase, a DNA template such as total cellular DNA, the four nucleotide triphosphates from which DNA is synthesized, a buffered solution containing appropriate ions, and two so-called oligonucleotide primers.
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From page 20... ...
These fragments can be detected rapidly and easily as a band of appropriate size after gel electrophoresis. PCR amplification -of DNA has been greatly simplified by the use of heat-resistant DNA polymerase found in Thermus aquaticus, bacteria native to hot springs, and through the development of program
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From page 21... ...
In many but not all situations, this technique is sufficiently sensitive to detect single nucleotide differences between the probe and the target nucleic acid. Although construction of RNA probes for DNA sequences has been simplified by PCR with primers that incorporate promoters for in vitro transcription of the fragment into RNA (9)
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From page 22... ...
Single base pair differences can apparently often produce sufficient changes in the overall three-dimensional structure of these tangles that a mutant fragment will migrate differently from a wild type fragment in a polyacrylamide gel. In practice, the technique works with fragments of DNA up to about 500 base pairs in size, but the precise conditions for gel electrophoresis (mostly concerning proper temperature)
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From page 23... ...
bY using an enzyme called reverse transcriptase Techniques for Analyzing Changes at the Protein Level All of the techniques used in clinical testing involve antibodies directed against the protein of interest. In one form of analysis called the Western blot, proteins isolated from tumor are separated by gel electrophoresis, transferred to a membrane, and identified by incubating the membrane with specific antibody.
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From page 24... ...
The principal limitation at present is that most genetic changes associated with oncogenesis cannot be assayed by using this technology.
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