Prevention of carryover contamination in the detection of βS and βC genes by polymerase chain reaction

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Abstract
As the polymerase chain reaction (PCR) process becomes a common tool in genetic diagnostic laboratories, prevention of carryover contamination from previous PCR amplifications has become an urgent topic. A PCR carryover prevention technique, utilizing deoxyuridine triphosphate (dUTP) and uracil DNA glycosylase (UDG), has been described recently. We report on its adaptation to a diagnostic system for detecting hemoglobin SS and SC diseases. Excellent amplification was achieved by increasing the dUTP and MgCl2 concentrations. dU-containing DNA could be analyzed by restriction endonucleases to distinguish the βS from βA gene using Ddel, but two other restriction enzymes, Bsu36l (replacement for Mstll by the manufacturer) and Cvnl, can no longer be used. The dU-containing PCR products retained their hybridization specificity with allele-specific oligonucleotide (ASO) probes for the βA, βS, and βC genes. The PCR carryover prevention technique is easy to use and takes only 20 additional minutes. It should be extremely useful to genetic diagnostic laboratories where PCR is repeated daily and carryover contamination may thus lead to misdiagnoses.