Abstract
Small variations, even single-nucleotide variants in nucleic acid sequences may exert significant influence in many diseases. The reliable detection and quantification of DNA can provide a variety of information that has significant clinical value, including disease risk assessment, screening, diagnosis, prognosis, the selection of therapies and monitoring. Due to the fact coupling efficiency of dNTPs and purification methodology can limit the chemical synthesis of long stranded probes. We demonstrated a PCR-based method to construct a long-stranded detection probe with high levels of selectivity via a strand displacement reaction with ssDNA target generates by asymmetric PCR. This detection probe was enzymatically generated from a double-stranded DNA duplex, containing a single-stranded active toehold domain. This approach was successfully implemented to genotype human glucose-6-phosphate dehydrogenase (G6PD) deficiency, focusing upon the c.1376G > T and c.1388G > A variants.
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