Biomarker anaylsis using scodaphoresis

Patent metadata

Field	Value
Publication number	US-9512477-B2
Application number	US-201313887060-A
Country	US
Kind code	B2
Filing date	May 3, 2013
Priority date	May 4, 2012
Publication date	Dec 6, 2016
Grant date	Dec 6, 2016

Abstract

The invention discloses methods and apparatus for characterizing trace nucleic acids that are biomarkers for disease. The methods and apparatus provide increased sensitivity to such trace nucleic acids, and allow analysis of nucleic acids present in a sample at only 0.01% of the wild-type sequences. The methods and apparatus are also designed for straightforward multiplexing, thus allowing pooling of clinical samples.

First claim

The invention claimed is:

1. A method of characterizing a biomarker in a sample, the method comprising:
providing a sample comprising a wild-type nucleic acid and a corresponding mutant nucleic acid that is a biomarker for a disease;
introducing a plurality of positive controls to the sample, the plurality of positive controls comprising a sequence identical to the mutant nucleic acid and a unique control sequence comprising a number of degenerate bases;
amplifying the mutant nucleic acid and the plurality of positive controls in the sample;
loading the amplified nucleic acid sample and the plurality of positive controls on a separation medium, the separation medium comprising an immobilized probe, the immobilized probe comprising a nucleic acid sequence complementary to the mutant nucleic acid;
enriching the amplified nucleic acid sample for the mutant nucleic acid and the plurality of positive controls over the wild type nucleic acid by applying a time-varying driving field and a time-varying mobility-varying field to the separation medium;
characterizing the enriched nucleic acid in the sample with a technique selected from nucleic acid sequencing, quantitative PCR (qPCR), mass spectrometry, and hybridization assay; and
quantifying abundance of the mutant nucleic acid in the provided sample using the plurality of positive controls.

2. The method of claim 1 , wherein characterizing the enriched nucleic acid comprises determining a sequence of the nucleic acid, determining an amount of the enriched nucleic acid as compared to another nucleic acid, or determining an absolute number of nucleic acid molecules in the sample.

3. The method of claim 1 , wherein nucleic acid sequencing is selected from Sanger sequencing, single molecule sequencing, nanopore-based sequencing, sequencing by synthesis, sequencing by ligation, pyrosequencing, sequencing by hydrogen ion release detection, ion semiconductor sequencing, and atomic force microscopy sequencing.

4. The method of claim 1 , wherein the nucleic acid is between about 20 and 100 nucleotides in length.

5. The method of claim 1 , wherein the sample is obtained from a tissue sample of a subject, a body fluid of a subject, a cell sample of a subject, or a stool sample of a subject.

6. The method of claim 5 , wherein the body fluid is selected from blood, a portion of whole blood, saliva, tears, sweat, sputum, urine, and nipple aspirate.

7. The method of claim 6 , wherein the portion of whole blood is blood plasma or cell-free nucleic acid.

8. The method of claim 5 , wherein the tissue sample is a formalin-fixed paraffin-embedded (FFPE) tissue sample.

9. The method of claim 1 , wherein amplifying comprises conducting between 1 and 10 cycles of PCR.

10. The method of claim 1 , wherein the amplified nucleic acid is cleaned prior to enrichment.

11. The method of claim 10 , wherein the amplified nucleic acid is cleaned using a commercial PCR clean-up column, by addition of an enzyme to specifically digest primers, by heat inactivation of enzymes remaining after amplification, or a combination thereof.

12. The method of claim 1 , wherein amplifying additionally comprises producing amplicons comprising barcodes.

13. The method of claim 1 , wherein applying a time-varying driving field and a time-varying mobility-varying field comprises applying two non-collinear time-dependent electric fields.

14. A method of characterizing a plurality of nucleic acids, comprising:
providing a sample comprising wild-type and mutant versions of a first nucleic acid and wild type and mutant versions of a second nucleic acid;
introducing a plurality of positive controls to the sample, each of the plurality of positive controls comprising a sequence identical to the mutant version of the first or second nucleic acid and a unique control sequence comprising a number of degenerate bases;
amplifying the mutant versions of the first and second nucleic acids and the plurality of controls in the sample;
loading the nucleic acid sample on a separation medium, the separation medium comprising:
a first immobilized probe comprising a nucleic acid sequence complementary to the mutant version of the first nucleic acid; and
a second immobilized probe comprising a nucleic acid sequence complementary to the mutant version of the second nucleic acid;

enriching the amplified nucleic acid sample for the mutant versions of the first and second nucleic acids and the plurality of positive controls by applying a time-varying driving field and a time-varying mobility-varying field to the separation medium;
characterizing the enriched first and second nucleic acids in the sample with a technique selected from nucleic acid sequencing, quantitative PCR (qPCR), mass spectrometry, and hybridization assay; and
quantifying abundance of the mutant versions of the first and second nucleic acids present in the provided sample using the plurality of positive controls.

15. The method of claim 14 , wherein the first and second nucleic acids comprise barcodes.

16. The method of claim 14 , wherein the first and second nucleic acids are PCR amplified with primers including barcode sequences.

17. The method of claim 16 , wherein the PCR is multiplexed.

18. The method of claim 14 , wherein the first nucleic acid and the second nucleic acids are from different subjects.

19. The method of claim 14 , wherein the first nucleic acid and the second nucleic acids are from the same subject.

20. The method of claim 1 , wherein the mutant nucleic acid is present at a 0.01% to 0.1% abundance relative to the wild-type nucleic acid in the sample.

21. The method of claim 14 , wherein at least one of the mutant versions of the first and second nucleic acids is present at a 0.01% to 0.1% abundance relative to the respective wild-type version.

Assignees

• Boreal Genomics Corp
• Univ British Columbia
• Boreal Genomics Inc

Inventors

• Marziali Andrea
• Wiggin Matthew
• Shibahara Gosuke
• Vysotskaia Valentina S

Classifications

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q1/6816 Primary

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title

• C12Q2565/125

  Electrophoretic separation · CPC title

• C12Q2565/607

  being a sensor, e.g. electrode · CPC title