SEQUENCING METHOD, APPARATUS, SYSTEM AND APPLICATION THEREOF

• US 20180195117A1
• Filed: 02/06/2018
• Published: 07/12/2018
• Est. Priority Date: 08/14/2015
• Status: Abandoned Application

• Current Assignee: Genemind Biosciences Company Limited
• Original Assignee: Direct Genomics Co. Ltd.
• Inventors: Ge, Liangjin, Gao, Yan, Deng, Liwei, Wu, Zengding, Cai, Jinsen, Ji, Daorui, Zeng, Lidong, Li, Gailing
• Application Number:
  US15/890,028
• Publication Number:
  US 20180195117A1
• Time in Patent Office: Days
• CPC Class Codes: C12M 1/34 Measuring or testing with condition measuring or sensing means, e.g. colony counters
• CPC Class Codes: C12M 1/36 including condition or time responsive control, e.g. automatically controlled fermentors controlling or regulating in general G05
• CPC Class Codes: C12M 1/38 Temperature-responsive control
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: G06T 2207/30072 Microarray; Biochip, DNA array; Well plate
• CPC Class Codes: G06T 7/0012 Biomedical image inspection

Abstract

A sequencing method comprising: (i) combining a template nucleic acid having a first optical detection label at the end with a primer to obtain a first complex; (ii) imaging the first complex to obtain a first image; (iii) mixing the first complex, polymerase, and one or more of nucleotides with a optical detection label to obtain an extension product by polymerization reaction; (iv) imaging the extension product to obtain a second image; (v) removing the cleavable group of the nucleotides with the optical detection label from the extension product to obtain a second complex; (vi) repeating the above steps (ii) to (v) once or more times to determine the template nucleic acid sequence. The sequencing method can achieve single molecule sequencing by capturing template nucleic acids through primers.

Claims

1. A single molecule sequencing method comprising:

(i) combining a template nucleic acid having a first optical detection label at the end with a primer to obtain a first complex, wherein the primer being attached to a surface of a substrate;

(ii) imaging the first complex to obtain a first image;

(iii) mixing the first complex, polymerase, and one or more of nucleotides with a second optical detection label and thus adding the one or more of nucleotides with the second optical detection label to the first complex by polymerization reaction, thereby obtaining an extension product, wherein the nucleotides with the second optical detection label comprises a second optical detection label, a cleavable group and a nucleotide that are sequentially connected;

(iv) imaging the extension product to obtain a second image;

(v) removing the cleavable group from the extension product to obtain a second complex; and

(vi) replacing the first complex with the second complex; and

repeating the above steps (ii) to (v) once or more times to determine the template nucleic acid sequence.

2. A method according to claim 1, wherein the first optical detection label is a non-light breakable label.

3. A method according to claim 1, wherein the primer is a nucleotide sequence having a polyT of 10-30 nt at the 5 ′

end.

4. A method according to claim 3, wherein the primer is attached to a substrate with surface-modified epoxy through an amino group modified at the 5 ′

end.

5. A method according to claim 1, wherein the optical detection label modified at the end of the template nucleic acid is a fluorescent label.

6. A method according to claim 1, wherein the cleavable group is a photo-cleavable group, a chemically-cleavable group and/or an enzyme-catalyzed cleavable group.

7. A method according to claim 1, wherein the step ii comprises:

imaging the first complex with different light sources to obtain a plurality of first images.

8. A method according to claim 1, wherein the step iv comprises:

imaging the extension product with different light sources to obtain a plurality of second images.

9. A method according to claim 1, wherein further comprises:

obtaining a template nucleic acid;

treating the template nucleic acid to obtain a template nucleic acid with a first optical detection label at its end.

10. A method according to claim 1, wherein the template nucleic acid is DNA and/or RNA.

11. A method according to claim 1, further comprises:

determining the template nucleic acid sequence based on the first image and the second image.

12. A method according to claim 11, wherein the determining the template nucleic acid sequence based on the first image and the second image comprises:

comparing the difference between the first images and/or the second images, and performing a first correction and/or a second correction on the first images and/or the second images, to obtain corrected first images and/or corrected second images.

13. A method according to claim 12, wherein the first correction is performed based on the first images and the second images in the same cycle of reaction, and

the second correction is performed based on between the first images or between the second images in the adjacent cycle of reaction, wherein from (ii) to (v) is defined as a cycle of reaction.

14. A single molecule sequencing apparatus comprising:

a combining module, for combining a template nucleic acid having a first optical detection label at the end with a primer to obtain a first complex, wherein the primer being attached to a surface of a substrate;

a first imaging module, for imaging the first complex from the combining module to obtain a first image;

a synthesizing module, for mixing the first complex from the combining module, polymerase, and one or more of nucleotides with a second optical detection label and thus adding the one or more of nucleotides with the second optical detection label to the first complex by polymerization reaction, thereby obtaining an extension product, wherein the nucleotides with the second optical detection label comprises a second optical detection label, a cleavable group and a nucleotide that are sequentially connected;

a second imaging module, for imaging the extension product from the synthesizing module to obtain a second image;

a cleaving module, for removing the cleavable group from the extension product from the synthesizing module to obtain a second complex; and

an iterating module, for replacing the first complex from the combining module with the second complex from the cleaving module, and entering in turn the first imaging module, the synthesizing module, the second imaging module and the cleaving module one or more times to determine the template nucleic acid sequence.

15. An apparatus according to claim 14, wherein the first imaging module is used for imaging the first complex with different light sources to obtain a plurality of first images.

16. An apparatus according to claim 14, wherein the second imaging module is used for imaging the extension product with different light sources to obtain a plurality of second images.

17. An apparatus according to claim 14, wherein further comprises image processing module, the image processing module is used for determining the template nucleic acid sequence based on the first image and the second image.

18. An apparatus according to claim 14, wherein the image processing module is used for comparing the difference between the first images and/or the second images, and performing a first correction and/or a second correction on the first images and/or the second images, to obtain a corrected first image and/or a corrected second image.

19. An apparatus according to claim 18, wherein

the first correction is performed based on the first image and the second image in the same cycle of reaction, and the second correction is performed based on between the first images or between the second images in the adjacent cycle of reaction, wherein entering the first imaging module, the synthesizing module, the second imaging module and the cleaving module in turn once is defined as a cycle of reaction.