Method for sequencing DNA and RNA by synthesis

• US 7,544,794 B1
• Filed: 03/11/2006
• Issued: 06/09/2009
• Est. Priority Date: 03/11/2005
• Status: Active Grant

• Current Assignee: Steven Albert Benner
• Original Assignee: Steven Albert Benner
• Inventors: Benner, Steven Albert
• Primary Examiner(s): Jiang; Shaojia Anna
• Assistant Examiner(s): OLSON, ERIC
• Application Number:
  US11/373,415
• Time in Patent Office: 1,186 Days
• Field of Search: None
• US Class Current: 536/27.61
• CPC Class Codes: C07H 19/044 Pyrrole radicals
• CPC Class Codes: C07H 19/167 with ribosyl as the saccharide radical
• CPC Class Codes: C07H 19/173 with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids

Abstract

This invention relates to the field of nucleic acid chemistry, more specifically to the field of compositions and processes that are nucleic acid analogs. More specifically, this invention relates to compositions that allow the sequencing of oligonucleotides by synthesis, and processes for sequencing by synthesis that exploit these compositions. Most specifically, the instant invention discloses compositions of matter that are 5′-triphosphates of ribo- and 2′-deoxyribonucleosides wherein the 3′-OH group is replaced by a 3′-ONHR group in the alpha configuration, wherein R is either a H or CH₃ group. Also disclosed are these triphosphates where the nucleobase carries, via a linker, a reporter groups, such as a fluorescent species that can be used in single- or multi-copy DNA sequencing, or a tag that can be visualized by ultramicroscopy. Also disclosed are processes that use these compositions to do sequencing by synthesis.

Claims

1. A composition of matter having the structure:

2. The compositions of claim 1, wherein said heterocycle is appended to a linker to which is appended a fluorescent group.

3. A process for extending a DNA primer by template-directed synthesis wherein said process comprises contacting said primer and said template with a DNA polymerase or reverse transcriptase and a 2′

-deoxynucleoside triphosphate wherein the 3′

-OH group of said triphosphate is blocked by a removable protecting group comprising an —

O—

N—

linkage and having fewer than three non-hydrogen atoms.

4. The process of claim 3 wherein said protecting group is selected from the group consisting of NH₂ and NH—

CH₃.

5. The process of claim 3 wherein said DNA polymerase comprises a mutant DNA polymerase where one of the phenylalanine or tyrosine residues in contact with the 2′

-deoxyribose ring is replaced by a histidine or a phenylalanine.

6. A composition of matter having the structure: