A non-PCR-based Invader® assay quantitatively detects single-copy genes in complex plant genomes

Abstract

An accurate determination of gene copy number is critical to the success of a molecular breeding program involving both transgenic and non-transgenic plants. In this paper, we have described the application of a non-PCR-based technology, Invader®*, for determination of gene copy number and zygosity in plants. A biplex assay format detected both a target gene and an endogenous reference gene simultaneously from the genomic DNA. The ratio between the signals of the two genes in relation to known copy number standards of the same target gene allowed copy number determination. The linear range of the Invader assay was 1–4 copies per genome, but it can be accurate over a larger copy number range depending on the assay conditions. This technique was utilized for screening plants carrying low transgene copy numbers from a large number of events generated by plant transformation, and shown to produce results comparable to that of Southern blots. We have also utilized this technique to screen thousands of field-grown plants for zygosity determinations and obtained data that was over 98% accurate, thus proving that this assay can be used to improve the efficiency of a breeding program. Overall, the Invader assays proved to be reproducible, specific, applicable to any gene sequence and amenable to high-throughput screening.