Automation of polymerase chain reaction tests to achieve acceptable contamination rates.

Since the invention of the polymerase chain reaction (PCR), >26 000 papers have been published that in some way utilizethis technique for DNA analysis. Clinical applications remain largely in the research laboratory setting, but the first US Food and Drug Administrationapproved PCR-based diagnostic kit for Chiamydia trachomatis is already commercially available, and the PCR technique holds great promise for future clinical use (1, 2). Because of the extreme sensitivity of the method, strict laboratory protocolisrecommended (3) to minimize riskof human error,which can lead to false-positive results through DNA carryovercontamination (4, 5). Additionally, several physical and chemical methods have been developed that can be used prophylactically to prevent contamination or to help correct existing contamination problems (6-11). Automation is another means of improving the reliability of PCR-based DNA diagnostics (12), and Kodak (13) and Roche are independently developing automated PCR instrumentation. Unfortunately, we are aware of no published information defining acceptable contamination rates for a PCR laboratory with automated or manual technology. We have automated PCR-based DNA amplification by using a Zymate II robotics system (Zymark, Hopkinton, MA) (14, 15). Reagent assembly, sample loading, operation of PTC-100 programmable thermal controllers (MJ Research, Watertown, MA) for 96-well U-bottom and Vbottom microtiter plates, and mixing of the amplicon with loading buffer for ethidium bromide gel electrophoresis are all automated with thisroboticssystem. The DNA extraction is performed in the same room as the robotics system by methods appropriate to the sample used. For example, to obtain DNA from formalin-fixed, paraffinembedded tissue, we used a quick extraction method (16, 17) involving deparaffinization in xylenes, followed by sonication to lyse the cells and digestion with proteinase K. DNA from other samples were extracted by the chloroform/phenol method, followed by precipitation with isopropyl alcohol, a wash with 70% ethanol, and resuspension in the PCR buffer (18). The amplified DNA was analyzed by agarose gel electrophoresis and photographed under ultraviolet transillumination of the ethidium bromide-stained gels. Over the 24 months of robotics operation (November 1992 through October 1994), we studied several diverse analytes, including cytomegalovirus XbaI-E DNA, ras and p.5,3 oncogenes, beta-globin, Helicobacter pylon-specific 16S rDNA, universal bacterialprimers utilizing16S rDNA, and Mycobacterium paratuberculosis 1S900. Samples analyzed included formalin-fixed, paraffin-embedded tissues; buffy coats; sewage; fermenter suspensions; and microbial cell cultures. An average of 35 reactions were performed per thermal-cycled experiment. To maximize the screen for contamination, we designed the study so that 21% of all reactions were negative controls (see Table 1). The only contamination occurred during the first 10 months of operation, and each event was corrected by the following working day.Because allcontaminationevents Table 1. Contamination ratesforPCR reactions performed by robotic apparatus.