Residual DNA in thermostable DNA polymerases - a cause of irritation in diagnostic PCR and microarray assays.
In a validation trial of a DNA microarray test for chlamydiae we repeatedly observed false-positive PCR amplicons from truly negative samples and non-template controls. Various PCR tests, microarray hybridization and DNA sequencing, revealed that residual Escherichia coli DNA from thermostable DNA polymerases was the cause of this cross-reaction. A subsequent survey showed that only five out of 23 commercial polymerases were free of E. coli DNA. When designing generic oligonucleotide sequences for PCR and PCR microarray-based assays one should be aware of such possible internal contamination, particularly when the target organism is E. coli.
Ehricht
Ralf
R
CLONDIAG chip technologies GmbH, Loebstedter Strasse 103-105, 07749 Jena, Germany. ralf@clondiag.com
Hotzel
Helmut
H
Sachse
Konrad
K
Slickers
Peter
P
D002693:Chlamydiaceae / Q000235:genetics / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D004866:Equipment Contamination; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification; D046228:Microarray Analysis / Q000592:standards*; D016133:Polymerase Chain Reaction / Q000592:standards*; D011933:Reagent Kits, Diagnostic / Q000382:microbiology*; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000737:chemistry; D013696:Temperature
D004269:DNA, Bacterial; D011933:Reagent Kits, Diagnostic; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase