Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction

The invasive signal amplification reaction has been previously developed fo r quantitative detection of nucleic acids and discrimination of single-nucl eotide polymorphisms. Here we describe a method that couples two invasive r eactions into a serial isothermal homogeneous assay using fluorescence reso nance energy transfer detection. The serial version of the assay generates more than 10(7) reporter molecules for each molecule of target DNA in a 4-h reaction; this sensitivity, coupled with the exquisite specificity of the reaction, is sufficient for direct detection of less than 1.000 target mole cules with no prior target amplification. Here we present a kinetic analysi s of the parameters affecting signal and background generation in the seria l invasive signal amplification reaction and describe a simple kinetic mode l of the assay. We demonstrate the ability of the assay to detect as few as 600 copies of the methylene tetrahydrofolate reductase gene in samples of human genomic DNA. We also demonstrate the ability of the assay to discrimi nate single base differences in this gene by using 20 ng of human genomic D NA.