Enzymological description of multitemplate PCR—Shrinking amplification bias by optimizing the polymerase–template ratio

摘要: Abstract Multitemplate polymerase chain reaction (PCR) is used for preparative and analytical applications in diagnostics research. Classical PCR qPCR are two basic setups with many possible experimental modifications. a method of choice to obtain enough material subsequent sophisticated such as construction libraries next-generation sequencing or high-throughput screening. Sequencing Single Nucleotide Primer Extension (SNuPE) employ one-strand synthesis represent distinct variant DNA synthesis. In all these applications, maintaining the initial ratio templates avoiding underestimation minority desired. Here, we demonstrate that different can amplify independently at low template concentrations (typical setups, which concentration usually several orders magnitude higher than concentration). However, rare be diluted an effort keep amplification exponential phase, biased by differences efficiency. Moreover, present more vulnerable stochastic events lead proportional changes product ratio, well incomplete leading chimera formation. These undesired effects compensated using highly processive polymerases high equal affinity primer–template complexes. Novel enhanced With increasing interest, system becomes deterministic. Nevertheless, marked deviation from independent occurs when total starts approach concentration. The complexes compete enzyme molecules, amount products grows arithmetically—the obey Michaelis–Menten kinetics. Synthesis multitemplate mixture run easily under detection limit conditions, although it would unequivocally detectable single assay. When fishing out variants, best should decrease both limits. possibility events, taken into account correctly interpret obtained data.