USE OF MANGANESE IN RT-PCR ELIMINATES PCR ARTIFACTS RESULTING FROM DNASE-I DIGESTION

The precise quantification of rare mRNA copies from intronless genes b y reverse transcription polymerase chain reaction (RT-PCR) requires th e complete removal of genomic DNA because discrimination of cDNA and D NA amplification products by differing sizes of PCR products is not po ssible. Elimination of DNA is achieved by treating the RNA sample with RNAse-free DNase I before RT-PCR. The lack of a PCR product from DNas e-treated RNA samples before RT is usually accepted as a proof of effi cient DNA destruction. However, this may vary depending on the metal c ofactor used in the DNase I cleavage. Treating DNA-contaminated RNA sa mples with DNase I and magnesium as a cofactor creates a negative PCR control after digestion without further RT. Paradoxically, after addit ional RT-PCR, the original intron-containing DNA fragment size may be produced again. In the presence of manganese as cofactor; RT-created D NA fragments do riot appear. This is because in the presence of mangan ese, DNase I cleaves both DNA strands at approximately the same site, yielding DNA fragments that are blunt-ended ed or that have protruding termini of only one or two nucleotides in length. However, overlappin g fragments with the potential to recombine are created by DNase diges tion with magnesium as cofactor: Because one cannot differentiate betw een a PCR signal produced by RNA and one produced by recombined DNA af ter DNase I digestion and RT: all such DNase I assays should be perfor med with manganese instead of magnesium.