Tuesday, November 14, 2006
162-1

Determination of Target Gene Copy Number in Plants Using QuantiGene Branched DNA Technology.

Cory Cui1, Jill Bryan1, Katherine Armstrong1, Thomas Patterson1, Joan Davies2, Botoul Maqsodi2, Jason Martin2, and Gary McMaster2. (1) Dow AgroSciences, 9330 Zionsville Road, Bldg. 306, Indianapolis, IN 46268-1054, (2) Panomics, Inc., 6519 Dumbarton Circle, Fremont, CA 94555

The world’s agriculture system has been revolutionized with the adoption of modern transgenic crops.  Molecular breeding and multiple gene stacking are playing major roles in crop improvement.  High throughput DNA manipulation and target gene tracking are pursuits in every biotech research lab and commercial agricultural company; however, a major bottleneck exists in DNA isolation. We describe a method that detects and quantifies the target gene in corn plants without DNA isolation or PCR amplification using branched DNA (bDNA) technology. 

 QuantiGene® Reagent System is a sandwich nucleic acid hybridization assay that provides a unique approach for DNA and RNA detection and quantification by amplifying the reporter signal using bDNA technology.  By measuring DNA or RNA directly from crude homogenates, the assay avoids variations or errors inherent to extraction and amplification of target sequences.  Lysed cells release target DNA or RNA in the presence of specific target probes and are captured by hybridization to a 96-well plate.  Signal amplification is performed by hybridization of the bDNA amplifier and label probe. Addition of chemiluminescence substrate yields a signal that is proportional to the amount of target sequence present in the sample.

 

With sensitivity to measure 10,000 target gene molecules, we demonstrate the utility of bDNA in tracking and determining the target gene copy number between various corn plants and through multiple generations.  Furthermore, we demonstrate that mechanical disruption of corn leaf punches gives a linear dose response to two single copy genes (Sus1 & Ivr1) and the multi-copy 18S ribosomal gene.  The bDNA assay has high precision, with typical CVs < 5%, and enables the quantitative measurement of gene copy directly from small amounts corn leaf tissue.  This experiment demonstrates that bDNA technology will be very useful for target gene tracking, transgene development, quick GMO testing, zygosity test, and molecular breeding.