Reagents for reversibly terminating primer extension

US8034923B1 · kind B1 · utility

Inventors

• Steven Albert Benner · Gainesville, US
• Daniel Hutter · Gainesville, US
• Nicole A Leal · Gainesville, US
• Fei Chen · Thạnh Trung, VN

Key dates

• Filing date: Mar 27, 2009
• Grant date: Oct 11, 2011
• Expiry date: May 11, 2030

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C07H21/00
• WIPO field: Organic fine chemistry
• WIPO sector: Chemistry

Abstract

Processes are disclosed that use 3′-reversibly terminated nucleoside triphosphates to analyze DNA for purposes other than sequencing using cyclic reversible termination. These processes are based on the unexpected ability of terminal transferase to accept these triphosphates as substrates, the unexpected ability of polymerases to add reversibly and irreversibly terminated triphosphates in competition with each other, the development of cleavage conditions to remove the terminating group rapidly, in high yield, and without substantial damage to the terminated oligonucleotide product, and the ability of reversibly terminated primer extension products to capture groups. The presently preferred embodiments of the disclosed processes use a triphosphate having its 3′-OH group blocked as a 3′-ONH2 group, which can be removed in buffered NaNO2 and use variants of Taq DNA polymerase, including one that has a replacement (L616A).