a 40 ml total starting volume with 30 nM substrate, 19.8 mM HEPES (pH 7.3), 1.8 mM b-ME and EDTA or metal at the indicated concentration. Reactions were dehydrated by SpeedVac without heating for 5 h, followed by heating of the dried pellet at 95 C for the indicated length of time. Dehydrated and/or heated reactions were resuspended in 40 ml H2O at room temperature for 5 min with periodic vor- texing. Reactions were puriﬁed via the Qiaquick Nucleotide Puriﬁcation Kit as discussed above for IN containing reac- tions. Analysis of reaction products via TaqMan qPCR or [g -32P]ATP labeled LRT F primer was performed as described above. RESULTS qPCR-based assay for disintegration We developed a qPCR assay to boost the sensitivity for detecting disintegration (Figure 1B). In previous studies, dis- integration assays were carried out using 32P end-labeled oligonucleotides, with the label attached to the nicked target strand, so that disintegration generated a longer strand (Figure 1A, star). The longer DNA product strand could then be quantiﬁed using PhosphorImager analysis. To increase the assay sensitivity, a substrate was designed composed of the four oligonucleotides indicated in Figure 1B, which annealed to form a DNA three-way junction. The Nucleic Acids Research, 2006, Vol. 34, No. 21 6119 substrate models a single HIV-1 LTR end inserted into a tar- get DNA. The target DNA has a 16 nt double-stranded region on either side of the LTR insertion and single-stranded exten- sions 50 and 30, which contain regions for the binding of pri- mers and a TaqMan probe. The transesteriﬁcation reaction involves attack of the free 30 hydroxyl on the phosphodiester between the LTR and target DNA segments, removing the LTR duplex and joining the target DNA to form a 97mer product. The 97mer product can be quantitated using TaqMan based qPCR. A single-stranded oligonucleotide matching the 97mer dis- integration product was synthesized and used to make a stan- dard curve for quantitation of PCR products (see Figure 2A). Ampliﬁcation of dilutions of the 97mer for standard curve generation yielded reproducible signals with correlation coefﬁcients >0.99. Our ﬁrst substrate design using all DNA oligonucleotides was not successful. The amounts of apparent disintegration product detected using the qPCR-based disintegration assay were roughly 1000-fold less than the amount detected in identical reactions using end-labeled substrates analyzed by gel electrophoresis (data not shown). As one possible improvement, we substituted the bottom DNA strand (Tbottom-32) with an RNA strand of identical length and sequence, based on the idea that the Tbottom-32 strand inhibited elongation during PCR, and an RNA strand could be degraded by addition of RNase. Much higher levels of Figure 2. Sensitivity of the PCR-based disintegration assay. (A) A standard curve was generated using 4-fold dilutions of the synthetic 97mer product oligonucleotide (Dis Full-97). The standard curve was linear and reproducible in the range tested (from 50 to 200 000 copies). (B) Disintegration reactions were carried out with dilutions of HIV-1 IN and analyzed in triplicate by qPCR. The IN reaction products were diluted by the factor shown. The threshold cycles for the IN reactions were compared to the standard curve for quantitation of product copies. (C) Disintegration reactions were carried out on a substrate with the Ttop-60 oligonucleotide 32P-labeled at the 50 end. Reactions were analyzed by 8% denaturing-polyacrylamide gel and PhosphorImager. (D) The total number product copies for reactions with radiolabeled substrate were calculated after measurement of percent product conversion at each IN concentration using ImageQuant software. Assay background was determined from the reaction with no IN added.