Abstract: The present disclosure provides methods and kits for ligation-coupled PCR. Methods for performing ligation and PCR and, optionally, enzymatic digestion in a single closed tube are provided. Methods and kits for splint-mediated primer assembly and ligation-coupled PCR are also provided.

Abstract: The present disclosure provides polynucleotide-based inhibitors for reversible activation of DNA polymerases. Use of lower Tm polynucleotide-based inhibitors allow PCR reaction assembly at room temperature while activating polymerase at higher PCR primer annealing temperatures, where the reversible nature of the inhibition additionally improves priming specificity during each PCR cycle. Additionally, temperature controlled inactivation of polymerase activity after PCR or other polymerase based enzymatic incubation eliminates a purification step when needed for compatibility with subsequent enzymatic incubations. For this application, the Tm of the polynucleotide-based inhibitor is higher than the desired reaction conditions of the subsequent enzymatic incubation.

Abstract: The present disclosure provides kits and methods for a targeted NGS library workflow comprising a multiplexed PCR for target amplification followed by an indexing PCR for incorporation of NGS adapter sequences. This workflow enables overlapping primer pairs in a single tube for contiguous coverage over target regions, while simultaneously preventing amplification of both primer dimers and undesirable mini-amplicons that result from overlapping primer pairs.