KAPA HiFi Uracil+

Site-directed mutagenesis is a powerful tool for protein engineering and the study of protein structure and function. Numerous methods of site-directed mutagenesis that exploit the primer extension technique have been developed. These methods generate heteroduplex species containing one mutated and one non-mutated strand; subsequent replication and segregation of these molecules inevitably results in a population heavily contaminated with wild-type DNA. Various methods that circumvent the replication of non-mutated DNA or that separate the two species have been developed, but these methods tend to be more difficult and lengthy. This application note describes a fast, simple, and efficient system for the introduction of specific mutations, insertions and/or deletions into DNA cloned in a double-stranded plasmid.

Using a number of case studies, this poster illustrates that engineered DNA polymerases are capable of providing better sequence coverage and are enabling novel and challenging NGS applications. The vast majority of PCR applications utilize a small number of wild-type DNA polymerases; improvements to existing applications and the development of novel PCR applications have primarily been realized via innovative changes to all of the reaction components except the polymerases themselves. Kapa Biosystems have engineered a new generation of improved DNA polymerases such as the KAPA HiFi. These are capable of robust amplification of diverse targets spanning a wide range of sizes and GC-content, and KAPA HiFi has demonstrated production of high yields with remarkably little amplification bias when used for NGS library amplification.

This application note provides a robust methodology for RRBS analysis using the streamlined chemistry of the KAPA Hyper Prep Kit and HiFi Uracil+ HotStart DNA Polymerase, and demonstrates its utility in accurately quantifying the DNA methylation status from sample-limiting conditions. Methyl-Seq is a powerful tool for understanding genome-wide methylation with base-pair resolution. Several strategies have been developed to understand the methylation status of a genome, including reduced representation bisulfite sequencing (RRBS). Bisulfite treatment results in a significant decrease in DNA input and quality; therefore, the construction of high-quality libraries and efficient downstream amplification is critical.