Genotyping Using Unlabeled Oligonucleotide Detection Probes
Deepika de Silva, Ph.D.
Method background
Homogenous PCR methods for genotyping require expensive fluorescently labeled oligonucleotide probes. We use a single 3'-blocked, unlabeled oligonucleotide probe and the saturating dye LCGreen Plus (Idaho Technology, Salt Lake City, UT) to generate melting curves characteristic of the genotype under the probe. Melting curves are generated and analyzed using the LightScanner Instrument System (Idaho Technology).
Genotyping with unlabeled probes is a homogeneous,
end-point assay. The probe is included in the PCR mix but is not consumed during
amplification. Genotyping is accomplished by monitoring the melting of probe-target
duplexes post-PCR. Key to the method is the use of one primer in excess, which
leads to the overproduction of the target strand. The method provides the same
specificity as current methods without the cost. Due to the equations in this
article, we have made a PDF
available for you to download.
