Prevention of carryover contamination in the detection of beta S and beta C genes by polymerase chain reaction.
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 beta S from beta A gene using Ddel, but two other restriction enzymes, Bsu361 (replacement for MstII by the manufacturer) and CvnI, can no longer be used. The dU-containing PCR products retained their hybridization specificity with allele-specific oligonucleotide (ASO) probes for the beta A, beta S, and beta C 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.
Wang
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X
Comprehensive Sickle Cell Center, Columbia University, New York, New York.
Chen
T
T
Kim
D
D
Piomelli
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S
D001483:Base Sequence; D001609:Beta-Globulins / Q000235:genetics*; D004247:DNA / Q000235:genetics; D045647:DNA Glycosylases; D004262:DNA Restriction Enzymes; D003856:Deoxyuracil Nucleotides; D005796:Genes / Q000235:genetics*; D006450:Hemoglobin SC Disease / Q000097:blood / Q000175:diagnosis; D006801:Humans; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases; D016133:Polymerase Chain Reaction / Q000379:methods; D051981:Uracil-DNA Glycosidase
D001609:Beta-Globulins; D003856:Deoxyuracil Nucleotides; C027078:deoxyuridine triphosphate; D004247:DNA; D004262:DNA Restriction Enzymes; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase