What is meant by polymerase fidelity? What applications require a high-fidelity polymerase?

The fidelity of a DNA polymerase refers to its ability to accurately replicate a template, or to add the correct nucleotides starting at the 3' end of the primer. The rate of base misincorporation is known as the error rate. PCR polymerases with proofreading activity possess 3'?5' exonuclease activity that can excise incorrectly incorporated nucleotides and replace them with the correct [...]

Which polymerases generate blunt ends versus A-overhangs?

-Enzymes in the PrimeSTAR series These enzymes exhibit substantial 3'?5' exonuclease activity and primarily generate amplification products with blunt ends. Therefore, we recommend using the Mighty Cloning Reagent Set (Blunt End) for blunt-end cloning. -Taq and Terra enzymes Takara Taq, Takara Ex Taq, Takara LA Taq, SpeedSTAR HS, EmeraldAmp, SapphireAmp Fast, and Terra PCR DNA polymerases [...]

What is touchdown PCR (TD-PCR) and when would I need to use it?

During the PCR denaturation step, all DNA molecules will become single stranded. When the temperature decreases for annealing, three types of duplexes can be formed: -Homoduplexes—annealing of complementary strands -Heteroduplexes—cross-hybridization of homologous sequences that may have partial homology -Duplexes between primers and template To achieve higher specificity, heteroduplex [...]

What is nested PCR?

Nested PCR is a method that involves re-amplification to improve PCR results. Nested PCR involves designing a new forward-nested (FN) or reverse-nested (RN) primer that is internal to the original primer and can pair with the original partner primer. A very small amount of the primary PCR product is used as a template for PCR with nested primers. Nested PCR frequently leads to improved yield of [...]

What factors are critical for multiplex PCR?

All primer pairs used in multiplex PCR should have similar priming efficiencies for their target DNA. This can be achieved by using primers with nearly identical optimum annealing temperatures. When designing primers, pay special attention the following parameters: -Homology with the target nucleic acid sequence -Length -GC content -Concentration -Primer homology (primers should not have [...]