QUANTITATIVE NUCLEASE PROTECTION ASSAY (QNPA) AND SEQUENCING (QNPS) IMPROVEMENTS

摘要

The present disclosure provides an improvement to quantitative Nuclease Protection Assay (qNPA) and quantitative Nuclease Protection Sequencing (qNPS) methods. The disclosed methods are improved in that they use nuclease protection probes that include 5'-end and/or 3'-end flanking sequences (NPPFs), the flanking sequences providing a universal hybridization and/or amplification sequence, and nucleic acid molecules comprising a sequence complementary to all or a portion of the flanking sequences (CFSs). Hybridization of the CFSs to the flanking sequences protects the flanking sequences from degradation during treatment with single-strand-specific nucleases, thus preserving elements in the flanking sequences for e.g., subsequent amplification, capture and/or analysis. The disclosed methods can be used to sequence or detect target nucleic acid molecules, such as those present in fixed or insoluble samples.