Rare DNA-sequence variants hold important clinical and biological information, but existing detection techniques are expensive, complex, allele-specific, or don’t allow for significant multiplexing. Here, we report a temperature-robust polymerase-chain-reaction method, which we term blocker displacement amplification (BDA), that selectively amplifies all sequence variants, including single-nucleotide variants (SNVs), within a roughly 20-nucleotide window by 1,000-fold over wild-type sequences. This allows for easy detection and quantitation of hundreds of potential variants originally at ≤0.1% in allele frequency. BDA is compatible with inexpensive thermocycler instrumentation and employs a rationally designed competitive hybridization reaction to achieve comparable enrichment performance across annealing temperatures ranging from 56 °C to 64 °C. To show the sequence generality of BDA, we demonstrate enrichment of 156 SNVs and the reliable detection of single-digit copies. We also show that the BDA detection of rare driver mutations in cell-free DNA samples extracted from the blood plasma of lung-cancer patients is highly consistent with deep sequencing using molecular lineage tags, with a receiver operator characteristic accuracy of 95%.

No abstract

Purification of oligonucleotides has traditionally relied on mobility-based separation methods. However, these are imperfect, biased, and difficult to scale high multiplex. Here, we present a method for simultaneous purification of many oligonucleotides that also normalizes concentrations. The method uses a rationally designed randomer capture probe to enrich for oligos with perfect 5′ sequences, based on the observation that synthesis errors are correlated: product molecules with one or more deletions in one region are also more likely to have deletions in other regions. Next-generation sequencing analysis of 64-plex 70 nt purification products show a median 78% purity, a significant improvement over polyacrylamide gel electrophoresis and high pressure liquid chromatography (60% median purity). Additionally, 89% of the oligo products are within a factor of 2 of the median concentration.

Type II phosphatidylinositol phosphate kinase (PIPKII) is an enzyme responsible for the synthesis of phosphatidylinositol-4,5-bisphosphate (PI-4,5-P(2)) from phosphatidylinositol-5-phosphate (PI-5-P). In this study, we demonstrate the presence of PIPKII alpha in bovine photoreceptor rod outer segments (ROS) and the involvement of tyrosine phosphorylation in the regulation of its activity. PIPKII activity in bovine ROS was verified by the preferential conversion of synthetic dipalmitoyl PI-5-P to PI-4,5-P(2), lack of effect of phosphatidic acid, inhibition by heparin, immunoreaction with an anti-PIPKII alpha antibody on Western blots, and immunocytochemical localization in bovine and rat ROS by anti-PIPKII alpha. Immunoprecipitates of bovine ROS with the anti-PIPKII alpha antibody possessed PIPK enzymatic activity and preferentially used PI-5-P as substrate for PI-4,5-P(2) biosynthesis. The activity of PIPKII was greatly increased under conditions favoring tyrosine phosphorylation in ROS, and PIPKII activity was immunoprecipitated with anti-phosphotyrosine (anti-PY) antibodies from tyrosine phosphorylated ROS. Preincubation of ROS with tyrosine kinase inhibitors almost abolished the kinase activity in the anti-PY immunoprecipitates. Immunoblot analysis showed that PIPKII alpha was present in anti-PY immunoprecipitates from phosphorylated ROS but not from nonphosphorylated controls. We conclude that PIPKII alpha is present in ROS and that its activity is regulated by tyrosine phosphorylation.