Digital analysis of nucleic acid modification

Patent metadata

Field	Value
Publication number	US-11901041-B2
Application number	US-201414505974-A
Country	US
Kind code	B2
Filing date	Oct 3, 2014
Priority date	Oct 4, 2013
Publication date	Feb 13, 2024
Grant date	Feb 13, 2024

Abstract

In certain aspects, methods of the invention involve performing modification state specific enzymatic reaction of nucleic acid in a sample, determining a value associated with efficiency of the modification state specific enzymatic reaction based on a control, determining an amount of target nucleic acid in the sample, and normalizing the amount of target nucleic acid based on the efficiency value. Based on the normalized amount of target nucleic acid, the method further includes determining whether the normalized amount of target nucleic acid is indicative of a condition.

First claim

What is claimed is:

1. A method of obtaining a normalized ratio between a fetal nucleic acid and a maternal reference nucleic acid in a mixture that provides a percent of the fetal nucleic acid in the mixture, the method comprising:
preparing a mixture comprising (i) a fetal nucleic acid comprising only recognition sequences that are unmethylated and is capable of cleaving by a restriction enzyme that cleaves only the recognition sequences that are unmethylated, (ii) a maternal reference nucleic acid comprising only the recognition sequences that are methylated and is not capable of cleaving by the restriction enzyme that cleaves only the recognition sequences that are unmethylated, and (iii) a known initial amount of control nucleic acid having only the recognition sequences that are unmethylated and is capable of cleaving by the restriction enzyme that cleaves only the recognition sequences that are unmethylated;
conducting a methylation-specific enzymatic reaction by exposing the mixture to the restriction enzyme that cleaves only the recognition sequences that are unmethylated;
partitioning the mixture into droplets after the conducting step;
measuring an amount of the fetal nucleic acid, an amount of the maternal reference nucleic acid, and an amount of the control nucleic acid in each of the droplets that remain intact by performing a digital polymerase chain reactions (PCR) in each of the droplets, wherein the measuring step includes the use of detectably labeled probes specifically hybridized to the fetal nucleic acid, the maternal reference nucleic acid, and the control nucleic acid;
determining an efficiency value for the methylation-specific enzymatic reaction based on the known initial amount of the control nucleic acid and the amount of the control nucleic acid determined in the measuring step; and
normalizing, using the efficiency value, the amount of the fetal nucleic acid and the amount of the maternal reference nucleic acid in the mixture, thereby obtaining the normalized ratio between the fetal nucleic acid and the maternal reference nucleic acid in the mixture that provides the percent of the fetal nucleic acid in the mixture.

2. The method of claim 1 , wherein the maternal reference nucleic acid is not a substrate for the methylation-specific reaction.

3. The method of claim 1 , wherein said measuring the amount of fetal nucleic acid includes counting a number of the droplets that include the fetal nucleic acid that remains intact after the digital PCRs and wherein said measuring the amount of control nucleic acid includes counting a number of the droplets that include the control nucleic acid that remains intact after the digital PCRs.

4. The method of claim 1 , wherein each of the fetal nucleic acid, the maternal reference nucleic acid, and the control nucleic acid comprises a promoter of a RASSFIA gene.

5. The method of claim 4 , further comprising detecting the amplified products in each of the droplets using fluorescent hydrolysis probes wherein the detectably labeled probes are the fluorescent hydrolysis probes.

6. The method of claim 4 , wherein the droplets are surrounded by an immiscible carrier fluid.

7. The method of claim 6 , wherein the immiscible carrier fluid comprises an oil.

8. The method of claim 7 , wherein the oil comprises a fluorosurfactant.

Assignees

• Bio Rad Laboratories Inc

Inventors

• Samuels Michael
• Olson Jeffrey
• Link Darren R

Classifications

• G16B20/10 Primary

  Ploidy or copy number detection · CPC title

• G16B20/00 Primary

  ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations · CPC title