Author:
Loan Thomas D.,Easton Christopher J.,Alissandratos Apostolos
Abstract
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 bacterial culture is enough to supply ~106 NAA reactions. Lyophilized lysate can be used after a single-step reconstitution, resulting overall in a greatly simplified workflow and a promising synthetic biology tool, in particular for applications such as diagnostics.
Publisher
Springer Science and Business Media LLC
Cited by
8 articles.
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