Substitution of 3′-Phosphate Cap with a Carbon-Based Blocker Reduces the Possibility of Fluorescence Resonance Energy Transfer Probe Failure in Real-Time PCR Assays

Abstract

During the last decade, research and clinical use of real-time PCR applications has continued to grow in importance (1). Many laboratories that use real-time PCR with fluorescent probes experience an unexplained loss of probe fluorescence at some stage, in particular with pairs of fluorescence resonance energy transfer (FRET) probes. Photobleaching is often assumed to be the cause. Structural integrity of the oligonucleotides is also a major factor, and its loss has been shown to correlate with repeated freeze–thaw cycles (2). Laboratories guard against these two problems by aliquoting probes and protecting them from light. Despite these precautions, inexplicable FRET probe failures are still observed. In one recent such case, we were able to determine an additional mechanism for FRET probe failure: loss of the phosphate cap from the 3′ end of a probe. To our knowledge, this has not been described previously. Our studies revealed that this may be a common and important problem, intrinsic to 3′-phosphate-blocking chemistry. We also found that alternative terminating groups may be a preferable option to 3′-phosphate blocking. A 3-nmol/L synthesis-scale LightCycler™ hybridization probe set was purchased from Idaho Technology Inc. Biochem in April 2003. As is common practice, the manufacturer produced a large-scale synthesis and, after shipping our order, archived the remainder for a possible future reorder. The first half of the batch (α-probe set) was sent immediately, whereas the second half (β-probe set) was stored lyophilized for 6 months at −20 °C and then shipped with our next order. Oligonucleotides from the first shipment were used in PCR reactions in a LightCycler with satisfactory results. PCR conditions were as follows: 1× LightCycler FastStart DNA Master Hybridization Probe Mix (Roche Diagnostics), MgCl2 (final concentration, 3.5 mM), 0.5 μM each of the forward (5′-GGCCTTTCTGAAGCAAG-3′) and reverse (5′-GACGATTTCTTATTTCACAGCTCC-3′) primers, 0.2 μM each of the donor …