DNA amplification with in situ nucleoside to dNTP synthesis, using a single recombinant cell lysate of E. coli.

ABSTRACT: Nucleic acid amplification (NAA) is a cornerstone of modern molecular and synthetic biology. Routine application by non-specialists, however, is hampered by difficulties with storing and handling the requisite labile and expensive reagents, such as deoxynucleoside triphosphates (dNTPs) and polymerases, and the complexity of protocols for their use. Here, a recombinant E. coli extract is reported that provides all the enzymes to support high-fidelity DNA amplification, and with labile dNTPs generated in situ from cheap and stable deoxynucleosides. Importantly, this is obtained from a single, engineered cell strain, through minimal processing, as a lysate capable of replacing the cold-stored commercial reagents in a typical PCR. This inexpensive preparation is highly active, as 1 L of bacter

SUBMITTER: Loan TD 

PROVIDER: S-EPMC6821818 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature