DNA sequencing

• US 10,584,377 B2
• Filed: 05/02/2013
• Issued: 03/10/2020
• Est. Priority Date: 05/02/2012
• Status: Active Grant

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Eshoo, Mark W.
• Primary Examiner(s): Chunduru, Suryaprabha
• Application Number:
  US14/398,271
• Publication Number:
  US 20150111762A1
• Time in Patent Office: 2,504 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/20 Sequence assembly

Abstract

Provided herein is technology relating to sequencing nucleic acids and particularly, but not exclusively, to methods, compositions, and systems for sequencing a nucleic acid using one or more labels and signal amplitude to distinguish bases.

Claims

1. A method for sequencing a target nucleic acid, the method comprising:

a) detecting a first amplitude of a first signal produced from a plurality of a first nucleotide base in a calibration oligonucleotide wherein a first known fraction of said plurality of a first nucleotide base in said calibration oligonucleotide is labeled with a fluorescent label;

b) detecting a second amplitude of a second signal from a plurality of a second nucleotide base in said calibration oligonucleotide wherein a second known fraction of said plurality of a second nucleotide base is labeled with said fluorescent label, wherein said first known fraction and said second known fraction are different known fractions, wherein said different known fractions differ between a plurality of different nucleotide bases in said calibration oligonucleotide, wherein said first amplitude is detectably different than said second amplitude, and wherein said calibration oligonucleotide comprises each of four different bases in known order;

c) detecting a first amplitude of a first signal produced from a plurality of a first nucleotide base in said target nucleic acid wherein a first known fraction of said plurality of a first nucleotide base is labeled with a fluorescent label;

d) detecting a second amplitude of a second signal from a plurality of a second nucleotide base in said target nucleic acid wherein a second known fraction of said plurality of a second nucleotide base is labeled with said fluorescent label wherein said first known fraction and said second known fraction are different known fractions, wherein said different known fractions differ between a plurality of different nucleotide bases in said target nucleic acid, wherein said first amplitude is detectably different than said second amplitude, and wherein the same nucleotide base in said calibration oligonucleotide and in said target nucleic acid is labeled with the same known fraction of said label;

e) associating said amplitude of said first signal with said first nucleotide base to identify said first nucleotide base in said calibration oligonucleotide and in said target nucleic acid; and

f) associating said amplitude of said second signal with said second nucleotide base to identify said second nucleotide base in said calibration oligonucleotide and in said target nucleic acid.

2. The method of claim 1 further comprising providing a first plurality of a first nucleotide base, a second plurality of a second nucleotide base, a third plurality of a third nucleotide base, and a fourth plurality of a fourth nucleotide base, wherein a first fraction of the first plurality of the first base is labeled with a label, a second fraction of the second plurality of the second nucleotide base is labeled with said label, a third fraction of the third plurality of the third nucleotide base is labeled with said label, and a fourth fraction of the fourth plurality of the fourth nucleotide base is labeled with said label.

3. The method of claim 2 wherein the first nucleotide base is A, the second nucleotide base is C, the third nucleotide base is G, and the fourth nucleotide base is T.

4. The method of claim 2 wherein the label is a fluorescent moiety.

5. The method of claim 1 further comprising providing a first plurality of a first nucleotide base, a second plurality of a second nucleotide base, a third plurality of a third nucleotide base, and a fourth plurality of a fourth nucleotide base, wherein a first fraction of the first plurality of the first base is labeled with a first label, a second fraction of the second plurality of the second nucleotide base is labeled with the first label, a third fraction of the third plurality of the third nucleotide base is labeled with a second label, and a fourth fraction of the fourth plurality of the fourth nucleotide base is labeled with the second label.

6. The method of claim 1 further comprising incorporating by polymerization the plurality of the nucleotide base into a plurality of a nucleic acid that is complementary to the target nucleic acid.

7. The method of claim 1 further comprising analyzing a dataset of ordered amplitudes to produce a nucleotide sequence of the target nucleic acid.