Preparation and Validation of PCR-generated Positive Controls for Diagnostic Dot Blotting

Abstract

Allele-specific oligonucleotides (ASOs) are small, single-stranded nucleotide polymers (∼18–20 bases in length) of diagnostic utility given their ability to hybridize to single-stranded DNA target molecules in a sequence-specific, temperature-dependent manner. Accordingly, two target molecules differing in composition by a single base can be distinguished by hybridization with an ASO that is complementary to one of the target molecules but noncomplementary to the other by a single base. The latter target produces a single basepair-mismatched double strand that dissociates at a lower washing temperature than the fully complementary, double-stranded molecule. Since first used to screen for beta-S globin mutations in sickle cell disease, ASOs have been widely used to detect known disease-causing mutations and disease-associated base substitutions (1)(2)(3). Diagnostic screening using ASOs for clinically relevant base substitutions (mutations/polymorphisms) frequently uses a dot blot format. PCR-amplified patient DNA is bound to a membrane, denatured to generate single-strand targets, and probed with both mutant and wild-type ASOs. Subsequent washing under optimized conditions denatures all but the entirely complementary double-stranded hybrids, which yield a positive signal. Dot blotting requires positive-control samples to monitor the sensitivity and specificity of each diagnostic blot. Given the rarity of some disease alleles, the acquisition of positive-control DNA is often rate-limiting in the establishment of …