Automated Polymerase Chain Reaction Research Articles

Automated polymerase chain reaction for papillomavirus screening of cervicovaginal lavages: Comparison with dot‐blot hybridization in a sexually transmitted diseases clinic population

The aim of the present study was to compare the recently developed polymerase chain reaction (PCR) technique with conventional dot-blot DNA hybridization for human papillomavirus (HPV) detection. Cells were collected by cervicovaginal lavage from a study group of 109 women attending a sexually transmitted diseases clinic. Using a machine that we developed for alternation of temperature cycles, HPV was detected in 51% of patients by PCR. By dot-blot hybridization, 44% of the patients were positive. Concordance of combined positive and negative results between PCR and dot blot was 69%. The greater sensitivity of PCR may have accounted for 19% of specimens that were PCR positive but dot-blot negative. Unexpectedly, however, 12% of specimens were dot-blot positive but negative by PCR, and several specimens were discordant for type of HPV. Both HPV DNA tests agreed with cytology in 41% of women, and in 33% cytology was negative in the face of positive PCR and dot blot. Concordance of cytology with just PCR was 59%, and only with dot blot was 56%. Cervicography agreed with both HPV DNA tests in 41% of patients, with PCR alone in 55%, and with dot blot alone in 58%. Biopsy results did not reveal a strong correlation between histopathological criteria of HPV infection and detection of HPV DNA by either PCR or dot-blot hybridization. Thus the present study has shown that PCR is a slightly more sensitive indicator of HPV infection than dot-blot hybridization. Agreement of HPV DNA results with conventional screening tests was not strong, an observation consistent with many comparative studies by others. In conclusion, PCR is slightly more sensitive than DNA hybridization for detection of HPV, it can be used in conjunction with specimen collection by gentle lavage of the cervicovaginal epithelium, and the possibility remains that it may prove suitable as a screening test.

The DNA polymerase from the archaebacterium Sulfolobus acidocaldarius: A thermophilic and thermoresistant enzyme which can perform automated polymerase chain reaction

A DNA polymerase purified from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius was used to perform automated DNA amplification at 70°C as well as site directed mutagenesis by Polymerase Chain Reaction (P.C.R.). The yield of amplification performed at optimum MgCl2 concentration for the Taq or the S. acidocaldarius DNA polymerase, for the same DNA target, was equivalent. The ability of S. acidocaldarius DNA polymerase to perform P.C.R. under less stringent requirement of MgCl2 concentration gives this enzyme a non-negligeable advantage over the Taq DNA polymerase.

How to build an inexpensive Cyclotherm instrument for automated polymerase chain reaction

The Cyclotherm instrument is a functionally fully equivalent but inexpensive alternative to commercial instruments for automated polymerase chain reaction (PCR). It can be rebuilt under conditions of a biochemical laboratory for less than $1000. A Peltier element is used for heating and cooling of the reaction vials and the temperature and timing of the PCR cycles are controlled by a BASIC program in a SHARP PC 1600 low cost computer.

Automated polymerase chain reaction in capillary tubes with hot air.

We describe a simple, compact, inexpensive thermal cycler that can be used for the polymerase chain reaction. Based on heat transfer with air to samples in sealed capillary tubes, the apparatus resembles a recirculating hair dryer. The temperature is regulated via thermocouple input to a programmable set-point process controller that provides proportional output to a solid state relay controlling a heating coil. For efficient cooling after the denaturation step, the controller activates a solenoid that opens a door to vent hot air and allows cool air to enter. Temperature-time profiles and amplification results approximate those obtained using water baths and microfuge tubes.