Recombinase-based amplification with substitute nucleotides

US9062336B1 · kind B1 · utility

Inventors

• Steven Albert Benner · Gainesville, US
• Nidhi Sharma · Pune, IN

Key dates

• Filing date: Mar 7, 2013
• Grant date: Jun 23, 2015
• Expiry date: Jul 18, 2033

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12Q1/6844
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

This invention covers methods for isothermal amplification of DNA. It is based on the unexpected discovery that primers having, at some positions, adenine substituted by 2-aminopurine or diaminopurine, guanine by inosine, thymine by 2-thiothymine, and cytosine by N4-ethylcytosine (“substituted primers”) were accepted by enzymes used in the standard recombinase polymerase assay (RPA). Further unexpected was the discovery that target nucleotides are efficiently amplified in an RPA-like process (hereinafter abbreviated as simply RPA) using substituted primers. RPA-like processes were also discovered to amplify target DNA with substituted primers tagged with oligonucleotides incorporating nucleotides from an artificially expanded genetic information system (AEGIS).