Methods for determining the amount of a nucleic acid of interest in an unprocessed sample

Patent metadata

Field	Value
Publication number	US-10590476-B2
Application number	US-201715711910-A
Country	US
Kind code	B2
Filing date	Sep 21, 2017
Priority date	Sep 23, 2016
Publication date	Mar 17, 2020
Grant date	Mar 17, 2020

Abstract

The present disclosure relates to methods for determining the amount or concentration of a nucleic acid of interest in an unprocessed sample by analyzing a processed sample and a reference sample with digital polymerase chain reaction (dPCR).

First claim

The invention claimed is:

1. A method for determining the amount or concentration of a nucleic acid of interest in an unprocessed sample, the method comprising the steps of:
a) providing an unprocessed sample suspected of containing the nucleic acid of interest and a reference sample known to contain a reference nucleic acid, which is different from the nucleic acid of interest;
b) combining the unprocessed sample with a defined amount of the reference sample, thereby obtaining a combined sample;
c) processing the combined sample, thereby obtaining a processed sample suitable for digital polymerase chain reaction (dPCR);
d) performing dPCR with the processed sample, thereby determining the amount or concentration of the nucleic acid of interest and the amount or concentration of the reference nucleic acid in the processed sample;
e) performing the dPCR with a defined amount of the reference sample, thereby determining the amount or concentration of the reference nucleic acid in the defined amount of the reference sample;
f) comparing the amount or concentration of the reference nucleic acid determined in step d) to that determined in step e), thereby determining the yield of the nucleic acid in step c); and
g) determining the amount or concentration of the nucleic acid of interest in the unprocessed sample based on the amount or concentration of the nucleic acid of interest in the processed sample determined in step d) and the yield determined in step f).

2. A method for determining the amount or concentration of a nudeic acid of interest in an unprocessed sample, the method comprising the steps of:
a) providing an unprocessed sample suspected of containing the nudeic acid of interest;
b) providing a reference sample known to contain a reference nucleic acid, which is different from the nucleic acid of interest;
c) processing the reference sample, thereby obtaining a processed reference sample suitable for dPCR;
d) performing the dPCR with the processed reference sample, thereby determining the amount or concentration of the reference nucleic acid in the processed reference sample;
e) performing the dPCR with a defined amount of unprocessed reference sample, thereby determining the amount or concentration of the reference nucleic acid in the defined amount of the unprocessed reference sample;
f) comparing the amount or concentration of the reference nucleic acid determined in step d) to that determined in step e), thereby determining the yield of the nucleic acid in step c);
g) processing the unprocessed sample, thereby obtaining a processed sample suitable for dPCR, wherein the processing steps c) and g) are identical;
h) performing the dPCR with the processed sample, thereby determining the amount or concentration of the nucleic acid of interest; and
i) determining the amount or concentration of the nucleic acid of interest in the unprocessed sample based on the amount or concentration of the nucleic acid of interest in the processed sample determined in step h) and the yield determined in step f).

3. The method of claim 2 , wherein performing steps a) and g) to i) is temporally, separated from performing steps b) to f).

4. The method of claim 1 , wherein (i) the amount or concentration of the reference nucleic acid in the reference sample is compared to a reference value, thereby controlling the reference sample; (ii) the amount or concentration of the reference nucleic acid in the reference sample is unknown or not predetermined; and/or (iii) the amount or concentration of the reference sample in step e) is identical to that in step b).

5. The method of claim 1 , wherein the reference nucleic acid has one or more of the following characteristics:
(i) is a nucleic acid selected from the group consisting of DNA, cDNA, RNA and a mixture thereof;
(ii) has the same primer binding site as the nucleic acid of interest;
(iii) has a primer binding site different from that of the nucleic acid of interest;
(iv) has a length in nucleic acids that differs from that of the nucleic acid of interest by at most 50%, at most 25%, at most 10% or at most 5%;
(v) has a sequence that is at least 50% identical, at least 60%, at least 70% or at least 80% identical to that of the nucleic acid of interest;
(vi) has a content of G and C that differs from that of the nucleic acid of interest by at most 50%, at most 25%, at most 10% or at most 5%; and
(vii) comprises a part that is not part of the nucleic acid of interest and that is used for detecting the reference nucleic acid.

6. The method of 5 , wherein the nucleic acid of interest has one or more of the following characteristics:
(i) is a nucleic acid selected from the group consisting of DNA, cDNA, RNA and a mixture thereof;
(ii) comprises a part that is not part of the reference nucleic acid and that is used for detecting the nucleic acid of interest; and
(iii) is indicative of a microorganism, a cell, a virus, a bacterium, a fungus, a mammal species, a genetic status or a disease.

7. The method of claim 1 , wherein the unprocessed sample has one or more of the following characteristics:
(i) has been obtained from a cell culture, a source suspected of being contaminated or a subject, wherein the subject is selected from the group consisting of a human, an animal and a plant; and
(ii) is selected from the group consisting of a body fluid, blood, blood plasma, blood serum, urine, bile, cerebrospinal fluid, a swab, a clinical specimen, an organ sample and a tissue sample.

8. The method of claim 1 , wherein the processing step comprises one or more of the following processes: dilution, lysis, centrifugation, extraction, precipitation, filtration, and purification.

9. The method of claim 1 , wherein dPCR is characterized by one or more of the following:
(i) is carried out in a liquid, in a gel, in an emulsion, in a droplet, in a microarray of miniaturized chambers, in a chamber of a microfluidic device, in a microwell plate, on a chip, in a capillary, on a nucleic acid binding surface or on a bead;
(ii) is carried out identically in at least 100 reaction areas; and
(iii) is carried out identically in at least 10,000 reaction areas.

10. The method of claim 1 , wherein steps d) and e) are carried out in the same dPCR run and/or on the same dPCR device.

11. The method of claim 1 , wherein dPCR comprises using one or more fluorescent probes, alone or in combination with a quencher, to detect the nucleic acid of interest and/or the reference nucleic acid.

12. The method of claim 11 , wherein the fluorescent probe comprises fluorescein, rhodamine, or cyanine.

13. The method of claim 1 , wherein the determining step comprises detecting a fluorescent signal.

14. The method of claim 1 , wherein the method further includes the use of an external control.

15. The method of claim 1 , wherein the method is used to diagnose the presence or absence of a disease, a pathogen, a rare genetic sequence, a rare mutation, a copy number variation or relative gene expression.

16. The method of claim 15 wherein the method is used to monitor disease progression, therapeutic response, and combinations thereof.

17. The method of claim 9 , wherein dPCR is carried out in a droplet.

Assignees

• Roche Molecular Systems Inc

Inventors

• Furlan Alan
• Zeder Michael

Classifications

• C12Q1/6851 Primary

  Quantitative amplification · CPC title

• C12Q1/686 Primary

  Polymerase chain reaction [PCR] · CPC title

• C12Q2531/113

  PCR · CPC title

• C12Q2545/101

  with an internal standard/control · CPC title

• C12Q1/6806 Primary

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