Effectiveness and limitations of uracil-DNA glycosylases in sensitive real-time PCR assays

mis. 2000. The continued evolution of two-hybrid screening approaches in yeast: how to outwit different preys with different baits. Gene 250:1-14.tions of high efficiency lithium acetate transformation of intact yeast cells using single-stranded nucleic acids as carrier. Yeast 7:253-263. 1999. Evidence that 3-phosphoinositide-dependent protein kinase-1 mediates phosphorylation of p70 S6 kinase in vivo at Thr-412 as well as Thr-252.tification of kinase-phosphatase signaling modules composed of p70 S6 kinase-protein phosphatase 2A (PP2A) and p21-activated ki-nase-PP2A.lecular cloning of CoA synthase. The missing link in CoA biosynthesis. S6 kinase complexes with and is activated by the Rho family G proteins Cdc42 and Rac1. PCR is sufficiently sensitive to detect single copy genes from single cells. This extreme sensitivity is also the basis of one of its potential problems; even a single product molecule from a previous amplification can lead to a false positive result. Substituting dUTP for dTTP during PCR and treating subsequent reactions with uracil-DNA glycosylase (UDG) prior to amplification is one strategy for limiting carryover contamination (1). However, total elimination of contaminants is not always accomplished using this technique, particularly where PCR product length is short (2), which is a common situation in real-time PCR assays. In addition, there is the possibility, particularly when the initial sample contains only one or a few target molecules, that inclusion of UDG may reduce amplification efficiency and thereby delay or prevent detection. As a first step toward implementing a general protocol for using UDG at the level of single copy genes in single cells, we investigated conditions for preventing contamination during amplification of a 133-bp segment within the multicopy testis-specific protein gene (TSPY) from single male lympho-cytes. Cell lysis and real-time PCR with molecular beacons were carried out as described previously (3), except that the extension step of thermal cycling was increased to 30 s and the dTTP was replaced with dUTP at a 3-fold higher concentration. The resulting PCR product contained 27 uracil residues in the sense strand and 28 uracil residues in the antisense strand. Initial experiments compared the efficiency of amplification in the presence of dUTP or dTTP in terms of the mean detection cycle (C T) value (i.e., the point at which the molecular beacon fluorescence intensity reaches a threshold of 200 U) and the final fluorescence after 45 cycles. Amplification plots are shown in Figure 1A. The mean C T values of 33.9 and 34.4 for dUTP and dTTP …