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US 10,590,476 B2Granted
Methods for determining the amount of a nucleic acid of interest in an unprocessed sample
AssigneeRoche Molecular Systems, Inc.
InventorsAlan Furlan, Michael Zeder
Life of this patent
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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).
Claims17
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).
5 more elements
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).
7 more elements
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.
1 more element
Dependencies are inferred from the claim text.