A New General Model for Predicting Melting Thermodynamics of Complementary and Mismatched B‑Form Duplexes Containing Locked Nucleic Acids: Application to Probe Design for Digital PCR Detection of Somatic Mutations

Advances in real-time polymerase chain reaction (PCR), as well as the emergence of digital PCR (dPCR) and useful modified nucleotide chemistries, including locked nucleic acids (LNAs), have created the potential to improve and expand clinical applications of PCR through their ability to better quantify and differentiate amplification products, but fully realizing this potential will require robust methods for designing dual-labeled hydrolysis probes and predicting their hybridization thermodynamics as a function of their sequence, chemistry, and template complementarity. We present here a nearest-neighbor thermodynamic model that accurately predicts the melting thermodynamics of a short oligonucleotide duplexed either to its perfect complement or to a template containing mismatched base pairs. The model may be applied to pure-DNA duplexes or to duplexes for which one strand contains any number and pattern of LNA substitutions. Perturbations to duplex stability arising from mismatched DNA:DNA or LNA:DNA base pairs are treated at the Gibbs energy level to maintain statistical significance in the regressed model parameters. This approach, when combined with the model’s accounting of the temperature dependencies of the melting enthalpy and entropy, permits accurate prediction of <i>T</i>_m values for pure-DNA homoduplexes or LNA-substituted heteroduplexes containing one or two independent mismatched base pairs. Terms accounting for changes in solution conditions and terminal addition of fluorescent dyes and quenchers are then introduced so that the model may be used to accurately predict and thereby tailor the <i>T</i>_m of a pure-DNA or LNA-substituted hydrolysis probe when duplexed either to its perfect-match template or to a template harboring a noncomplementary base. The model, which builds on classic nearest-neighbor thermodynamics, should therefore be of use to clinicians and biologists who require probes that distinguish and quantify two closely related alleles in either a quantitative PCR or dPCR assay. This potential is demonstrated by using the model to design allele-specific probes that completely discriminate and quantify clinically relevant mutant alleles (<i>BRAF</i> V600E and <i>KIT</i> D816V) in a dPCR assay

Initial Diagnosis of <i>ALK</i>-Positive Non-Small-Cell Lung Cancer Based on Analysis of <i>ALK</i> Status Utilizing Droplet Digital PCR

We describe a novel droplet digital PCR (ddPCR) assay capable of detecting genomic alterations associated with inversion translocations. It is applied here to detection of rearrangements in the anaplastic lymphoma kinase (<i>ALK</i>) gene associated with <i>ALK</i>-positive non-small-cell lung cancer (NSCLC). NSCLC patients may carry a nonreciprocal translocation on human chromosome 2, in which synchronized double stranded breaks (DSB) within the echinoderm microtubule-associated protein-like 4 (<i>EML4</i>) gene and <i>ALK</i> lead to an inversion of genetic material that forms the non-natural gene fusion <i>EML4-ALK</i> encoding a constitutively active tyrosine kinase that is associated with 3 to 7% of all NSCLCs. Detection of <i>ALK</i> rearrangements is currently achieved in clinics through direct visualization via a fluorescent <i>in situ</i> hybridization (FISH) assay, which can detect those rearrangements to a limit of detection (LOD) of ca. 15%. We show that the ddPCR assay presented here provides a LOD of 0.25% at lower cost and with faster turnaround times