Real-time sequencing method for single molecule

• US 9,540,688 B2
• Filed: 12/11/2014
• Issued: 01/10/2017
• Est. Priority Date: 02/19/2014
• Status: Active Grant

• Current Assignee: Personal Genomics, Inc.
• Original Assignee: Personal Genomics, Inc.
• Inventors: Fu, Tsu-Ju, Chang, Shang-Chia, Fann, Jenn-Yeh
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/566,715
• Publication Number:
  US 20150232928A1
• Time in Patent Office: 761 Days
• Field of Search: 435/6.1, 435/91.1, 435/91.2
• US Class Current: 1/1
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

This invention is to describe a method for the determination of a nucleic acid sequence, in which it involves an enzyme, an enzyme complex or plural number of enzymes with more than one enzymatic activity and a set of nucleotide analogs, to achieve high signal readout accuracy in nucleic acid sequencing by making each signal to have a long signaling time span which allows a higher signal clarity.

Claims

1. A method of using nucleotide analogs to determine a nucleotide sequence of a target nucleic acid, comprising the steps of:

(a) providing a reaction complex comprising a target nucleic acid, a primer nucleic acid comprising a sequence which is complementary to a region of the target nucleic acid, and a nucleic acid-polymerizing enzyme, enzyme complex, or enzyme mixture that possesses a 5′

to 3′

polymerization activity and a 3′

catalytic editing activity;

(b) contacting the reaction complex with nucleotide analogs, wherein each nucleotide analog comprises a base moiety and at least one label moiety comprising a photo-detectable label, wherein the photo-detectable label is connected to a 3′

position of a sugar of the nucleotide analog via a coupler group, and the at least one label moiety includes a linker group selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, polyethylene glycol, ester, sulfonyl or a combination thereof, connecting the photo-detectable label to the coupler group, and the nucleotide analog has a structure as shown in Formula I or Formula II;

4. The method of claim 1, wherein the photo-detectable label is a fluorophore or a fluorescent dye.

7. The method of claim 1, wherein the nucleotide analog has 3-12 phosphate groups at the 5′

-end of the nucleotide analog.

2. A method of using nucleotide analogs to determine a nucleotide sequence of a target nucleic acid, comprising the steps of:

(a) providing a reaction complex comprising a target nucleic acid, a primer nucleic acid comprising a sequence which is complementary to a region of the target nucleic acid, and a nucleic acid-polymerizing enzyme, enzyme complex, or enzyme mixture that possesses a 5′

to 3′

polymerization activity and a 3′

catalytic editing activity;

(b) contacting the reaction complex with nucleotide analogs, wherein each nucleotide analog comprises a base moiety and at least one label moiety comprising a photo-detectable label, wherein the photo-detectable label is connected to a 3′

position of a sugar of the nucleotide analog via a coupler group, and the at least one label moiety includes a linker group selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, polyethylene glycol, ester, sulfonyl or a combination thereof, connecting the photo-detectable label to the coupler group, and the nucleotide analog has a structure as shown in Formula XVI;

5. The method of claim 2, wherein the nucleotide analog has 3-12 phosphate groups at the 5′

-end of the nucleotide analog.

8. The method of claim 2, wherein the photo-detectable label is a fluorophore or a fluorescent dye.

3. A method of using nucleotide analogs to determine a nucleotide sequence of a target nucleic acid, comprising the steps of:

(a) providing a reaction complex comprising a target nucleic acid, a primer nucleic acid comprising a sequence which is complementary to a region of the target nucleic acid, and a nucleic acid-polymerizing enzyme, enzyme complex, or enzyme mixture that possesses a 5′

to 3′

polymerization activity and a 3′

catalytic editing activity;

(b) contacting the reaction complex with nucleotide analogs, wherein each nucleotide analog comprises a base moiety and at least one label moiety comprising a photo-detectable label, wherein the photo-detectable label is connected to a 3′

position of a sugar of the nucleotide analog via a coupler group, and the at least one label moiety includes a linker group selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, polyethylene glycol, ester, sulfonyl or a combination thereof, connecting the photo-detectable label to the coupler group, and the nucleotide analog has a structure as shown in Formula IA or Formula IIA;

6. The method of claim 3, wherein the nucleotide analog has 3-12 phosphate groups at the 5′

-end of the nucleotide analog.

9. The method of claim 3, wherein the photo-detectable label is a fluorophore or a fluorescent dye.