Abstract: A method for extracting DNA from a specimen is provided which is cost-efficient, nontoxic to laboratory workers and is automation-compatible to meet high-throughput requirements in newborn screening and other nucleic acid applications. Methanol is added and evaporated at a high temperature to ensure the heme and other large proteins bind to the filter paper thus preventing them from going into solution during extraction and inhibiting later PCR reactions. A buffer and salt concentration is then added to each specimen to continue to bind the heme protein to the filter paper when the DNA is extracted from the filter paper at an optimal pH. The plate is then heated to release the DNA into the buffer without releasing excess heme protein which may inhibit PCR reactions.

Abstract: What is disclosed is a system and method utilizing an automation system for high throughput DNA extraction and PCR setup, a conventional thermal cycler, and a LIGHTTYPER™ instrument for post-PCR melting temperature analysis for beta-globin mutations. Melting temperature analysis is achieved through fluorescent resonance energy transfer (FRET) reaction using the LIGHTTYPER™ instrument. The assay is designed to simultaneously detect three common beta-globin mutations, S(A173T), C(G172A), and E(G232A), and can identify any of the eight possible genotypes in a single reaction: AA, AE, EE, AS, SC, SS, AC, and CC (A represents wild type allele).