Quantitative PCR-ELAHA for the Determination of Retroviral Vector Transduction Efficiency  Ian M. Mackay, Pat Metharom, Theo P. Sloots, Ming Q. Wei  Molecular Therapy  Volume 3, Issue 5, Pages 801-808 (May 2001) DOI: 10.1006/mthe.2001.0320 Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 1 Construction of synthetic controls, pWSneo/pWSgfp and pISneo/pISgfp. Deletion of the wild-type probe binding sequences and subsequent introduction of unrelated sequences to produce the competitive template that provides the Q-PRB binding site, resulting in the internal standard (IS), are diagrammed. Mutated probe binding regions are shown on pISneo/pISgfp (▪) and neo-PRB/gfp- PRB binding sequences (□). Molecular Therapy 2001 3, 801-808DOI: (10.1006/mthe.2001.0320) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 2 Schematic representation of principle behind the ELAHA. Each sample's amplicon is diluted into tubes containing either the wild-type oligoprobe (neo-PRB/gfp-PRB) or the internal standard oligoprobe (Q-PRB) and hybridized by heat denaturation and rapid cooling under simple buffering conditions. The complex is bound to the plate and then incubated with an enzyme-conjugated antibody directed toward the DIG–11-dUTP moieties. Captured complexes can be visualized by a color that develops in proportion to the bound amplicon, after the addition of a substrate and chromogen. Molecular Therapy 2001 3, 801-808DOI: (10.1006/mthe.2001.0320) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 3 Determination of the dynamic range of the neo and gfp PCR. Samples were taken every 5-10 cycles from PCR using pISneo or pISgfp as template. Data were plotted as the cycle number at which amplicon first became positive by ELAHA vs the log10 of template starting copies. Molecular Therapy 2001 3, 801-808DOI: (10.1006/mthe.2001.0320) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 4 Sensitivity of ELAHA versus gel electrophoresis. pWSneo amplicon was titrated in 1× SSC and equal volumes were detected by agarose gel electrophoresis (inset) and ELAHA. Lanes 1–8 of inset, 10 μl of 1:2-1:256 dilutions of amplicon. Molecular Therapy 2001 3, 801-808DOI: (10.1006/mthe.2001.0320) Copyright © 2001 American Society for Gene Therapy Terms and Conditions