QuantiTect Multiplex PCR Kits

Dilutions of cDNA template (100 ng, 11.11 ng, 1.23 ng, and 0.14 ng) were analyzed by triplex PCR (colored curves) and by singleplex PCR (gray curves) on the iCycler iQ. TaqMan probes labeled with FAM, HEX, or Cyanine 670 dye were used. (Data kindly provided by the University of Minnesota, Minneapolis, MN, USA.)|GAPDH, 18S, HSP, and H28S sequences were amplified either together in 4-plex PCR or in singleplex PCRs on the Rotor-Gene 3000 system using the QuantiTect Multiplex PCR NoROX Kit. The templates were 100, 10, 1, and 0.1 ng of human leukocyte cDNA. Reactions were performed in triplicate. TaqMan probes labeled with FAM, HEX, Texas Red, or Cyanine 670 dye were used. 4-plex PCR and singleplex PCR data are displayed on the amplification plots, with the curves for the singleplex PCRs colored in gray. The plots show reliable multiplex quantification over a wide range of template amounts as well as good reproducibility within each set of triplicates. GAPDH: glyceraldehyde-3-phosphate dehydrogenase; 18S: 18S rRNA; HSP: a heat shock protein; H28S: 28S rRNA.|Three sequences (CSBG, GAPDH, and HSP) were amplified either together in triplex PCR or in singleplex PCRs on the LightCycler 2.0 system using the QuantiTect Multiplex PCR NoROX Kit. Three template mixes with varying amounts of human genomic DNA (for CSBG amplification), linearized plasmid (for GAPDH amplification), and human leukocyte cDNA (for HSP amplification) were prepared. Reactions were performed in duplicate. TaqMan probes labeled with FAM, HEX, or Texas Red dye were used. The amplification plot shows detection of CSBG in the triplex PCRs (Blue: 1400 copies; Green: 140 copies; Red: 14 copies).|[A] Duplex PCR was performed on the ABI PRISM 7700 using either the QuantiTect Multiplex PCR Kit or a kit from Supplier A_II. Variable amounts of t(8;14) translocation sequence (50,000, 5000, 500, 50, or 10 copies) were coamplified with a constant amount of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) sequence (10⁷ copies). The t(8;14) translocation sequence was amplified from Ramos cell line genomic DNA using a FAM labeled TaqMan probe. The GAPDH sequence was amplified from a plasmid containing GAPDH cDNA sequence using a HEX labeled TaqMan probe. In contrast to the kit from Supplier A_II, the QuantiTect Multiplex PCR Kit reliably quantified high to low amounts of t(8;14) translocation sequence (down to 10 copies, indicated with an arrow) in the presence of a high amount (10⁷ copies) of GAPDH sequence. [B] The procedure described in [A] was repeated, with the exception that the target sequences were amplified separately in singleplex PCRs.|QuantiTect Multiplex PCR Kits provide a simple procedure for quantitative, multiplex, real-time PCR. In contrast to current methods, the kits eliminate the need for optimization of the concentrations of primers, Mg²⁺, and Taq DNA polymerase, even for difficult assays (e.g., assays in which the copy number of the target gene is much smaller than that for the reference gene).|[A] NH₄⁺ ions prevent nonspecific primers from annealing to the template. [B] Synthetic Factor MP, an innovative PCR additive, increases the local concentration of primers at the template. Together with K⁺ and other cations, synthetic Factor MP stabilizes specifically bound primers, allowing efficient primer extension by HotStarTaq DNA Polymerase.|