Unnatural polymerase substrates that can sustain enzymatic synthesis of double stranded nucleic acids from a nucleic acid template and methods of use

US8058414B2 · kind B2 · utility

Assignee

• Life Technologies Corporation · US

Inventors

• Steven M. Menchen · Fremont, US
• Barnett Rosenblum · San Jose, US
• Paul Kenney · Sunnyvale, US
• Shoeb Khan · Durham, US
• Zhaochun Ma · Sunnyvale, US
• Jer-Kang Chen · Palo Alto, US
• Joe Y. L. Lam · Castro Valley, US
• Boli HUANG · Palo Alto, US

Key dates

• Filing date: Jan 16, 2009
• Grant date: Nov 15, 2011
• Expiry date: Jan 16, 2029

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12Q1/6874
• WIPO field: Organic fine chemistry
• WIPO sector: Chemistry

Abstract

Nucleotide analogs that can sustain the enzymatic synthesis of double-stranded nucleic acid from a nucleic template are described. The nucleotide analogs include: (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine, uracil and their analogs; (ii) a label attached to the base or analog of the base via a cleavable linker; (iii) a deoxyribose; and (iv) one or more phosphate groups. The linker and/or the label inhibits template directed polymerase incorporation of a further nucleotide substrate onto an extended primer strand. In addition, cleavage of the linker leaves a residue attached to the base which is not present in the natural nucleotide and which does not inhibit extension of the primer strand. The nucleotide analogs can therefore be used as reversible terminators in sequencing by synthesis methods without blocking the 3′ hydroxyl group. Methods of sequencing DNA using the substrates are also described.