Principle PRONTO® is a rapid, accurate and user-friendly assay for detecting Single Nucleotide Polymorphisms (SNPs) in DNA sequences. This post-amplification mutation detection system utilizes a single nucleotide primer extension assay. It benefits from the high specificity with which DNA polymerase incorporates nucleotides into the elongating strand. In the complete genotyping format of the PRONTO® system each mutation site is tested in two wells of a 96-well microtiter plate. Every sample is separately and internally controlled for each mutation tested. Results are clearly determined visually as well as analyzed by a standard ELISA reader. 1. DNA Extraction Pronto Diagnostics' DNA Extraction kit features a rapid, single test-tube procedure for isolating DNA from whole blood that is ready for direct use in DNA amplification reactions. Other methods that isolate DNA from whole blood have been validated for use with the PRONTO® kits. 2. Template Amplification The PRONTO® kits function over a range of template DNA concentrations (50-300 ng/ μl) and with validated DNA polymerases. The targeted gene sequences are simultaneously amplified, to about 106, using a multiplex DNA Amplification Mix. Amplification of target DNA to visible levels (10-100 ng/ μl) on an EtBr-stained gel is recommended. 3. Post-Amplification Treatment This reaction inactivates free nucleotides, which remain after the amplification step. 4. Primer Extension DNA polymerase extends a 5' FITC-labeled primer with a single biotinylated nucleotide, which complements the nucleotide at the tested site. Every reaction utilizes a different complementary bio-dNTP for the normal and the mutant alleles. Each mutation is tested in two wells: one for the mutant allele and one for the wild-type allele. The amplified DNA is dispensed into a 96-well thermowell plate using a multichannel pipette. The plate is placed in a thermocycler, where a short primer extension reaction takes place. 5. Detection by ELISA Reaction products are diluted and transferred to a streptavidin coated 96-well ELISA plate. Following a short incubation period, unbound primers are washed away and an HRP anti-FITC conjugate is added. The peroxidase reaction, that takes place in the presence of the HRP substrate, results in the appearance of a deep blue color in every positive well. 6. Visual Genotype Assignment For every mutation site tested, at least one of the wells should develop a deep blue color. Otherwise, results are invalid. 7. Interpretation of Results Numerical results can be obtained using an ELISA reader. Optical density (OD) readings are analyzed according to standard cut-off and mutant/wild-type ratio values.   PRONTO® Procedure Stage Assay Time* Template Amplification 90 minutes Post-Amplification Treatment 40 minutes Primer Extension 20 minutes Detection 30 minutes Total 3 Hours * Assay time may vary according to the type of thermocycler used.