Abstract
In this study, multiple displacement amplification (MDA) was used for transgene detection, and proved to effectively improve the sensitivity of most fluorescence polymerase chain (PCR). Five events of genetically modified plants were chosen for detection in different plant species by MDA and real-time PCRs. Individual primer/probe systems based on each transgene target were used. The extracted DNA of each sample was diluted and used as a template for MDA. All extracted DNA and diluted mdaDNA were detected by real-time PCR. The results were evaluated with respect to DNA quantity, DNA purity and difference of Ct values of the isogenetic DNA extract before and after MDA (D-values). The MDA amplifying effects were largely different among genes and plants. Organelle DNA has more copies than genomic DNA by MDA. Key words: Multiple displacement amplification, genetically modified organisms, detection, events.
Highlights
Multiple displacement amplification (MDA) which is widely used for whole genome amplification employs a bacteriophage φ29 DNA polymerase together with exonucleaseresistant degenerate primers to amplify DNA isothermally at 30°C (Dean et al, 2002; Lage et al, 2003)
Real-time PCR (RT-PCR), Southern blot analysis, conventional PCR and real-time PCR and transgene detection studies have been used to describe the utility of mdaDNA for generation of representative DNA (Hosono et al, 2003; Tengs et al, 2007)
The MDA of single cells for preimplantation genetic diagnosis was successfully used for analyzing Duchenne muscular dystrophy (Ren et al, 2009)
Summary
Multiple displacement amplification (MDA) which is widely used for whole genome amplification employs a bacteriophage φ29 DNA polymerase together with exonucleaseresistant degenerate primers to amplify DNA isothermally at 30°C (Dean et al, 2002; Lage et al, 2003). MDA has been used for accurate whole genome amplification from single cells and spores (Woyke et al, 2011). UV irradiation of MDA reagents effectively eliminates the exogenous DNA from whole genome amplification reagents for single cell sequencing and analysis (Woyke et al, 2011). MDA of gDNA from single spores of a rust fungus Puccinia striiformis can be used in molecular genetic analysis of the wheat yellow rust fungus (Wang et al, 2009).
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