PubMed · 7832901
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S M Crowther, J F Burke, S M Thomas. 1994-11-24. Polymerase patent problems.. https://doi.org/10.1038/372312a0
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To demonstrate the instability of microsatellite sequences, which have been linked to DNA mismatch repair deficiency and indicate a high risk of carcinogenesis, polymerase chain reaction (PCR) and electrophoretic analysis have been used. However, the electrophoretic profiles of PCR-amplified microsatellite sequences are often too complicated, and the conventional method using autoradiography has critical problems in detection characteristics and migration accuracy. First, we made use of fluorescence-labeled PCR and laser scanning to detect bands quantitatively. Second, we characterized Taq polymerase-dependent modification of the amplified microsatellite sequences and optimized the electrophoretic profiles by enzymatic modification with T4 DNA polymerase. Third, we developed a dual fluorescence co-electrophoresis system, in which both samples derived from cancer and normal tissues are electrophoresed in the same lane, in order to minimize migration errors. Furthermore, we classified all of the observed patterns of microsatellite alteration and set up new criteria for assessing microsatellite instability. Using the system developed here and the criteria we proposed, a precise judgment can be made and rates of positivity in various human malignancies may be corrected.
Three highly sensitive reverse transcriptase (RT) assays were recently published that are at least one million times more sensitive than conventional RT assays. These assays derive their high sensitivities through the ability to amplify the complementary DNA (cDNA) product of the RT reaction by the polymerase chain reaction (PCR). We describe a modified PCR-based RT (PBRT) assay that retains the high sensitivities of the original assays while reducing their inherent background signals. The background signal of the PBRT assay was found to be due to an intrinsic RNA-dependent DNA polymerase activity of the Taq DNA polymerase, the enzyme used for the PCR. It could be eliminated by inserting a ribonuclease digestion step prior to amplifying the cDNA product of the RT reaction by PCR and by using a thermostable DNA polymerase identified as having reduced RNA-dependent DNA polymerase activity. Comparable results were obtained using three RNA templates with two purified RT enzymes. This modified assay is capable of detecting reliably between 10 and 100 molecules of RT, which is equivalent to between 1 and 10 retrovirus particles.