Methods for analyzing DNA

• US 10,724,082 B2
• Filed: 12/08/2017
• Issued: 07/28/2020
• Est. Priority Date: 10/22/2012
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Samuels, Michael
• Primary Examiner(s): Kim, Young J
• Application Number:
  US15/835,697
• Publication Number:
  US 20180100185A1
• Time in Patent Office: 963 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2535/131 Allele specific probes
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

The invention generally relates to methods for increasing the amount of DNA available for analysis when using partitioned samples and parallel processing. For example, double-stranded DNA can be dissociated into two single-stranded components, and the single strands partitioned into different droplets prior to analysis. The disclosed methods are useful for performing digital PCR analysis on samples where the target DNA is not in abundance, for example when the sample originates from a body fluid or an FFPE sample.

Claims

1. A method for detecting DNA in a sample, the method comprising:

denaturing a double-stranded DNA in the sample into a first strand and a second strand;

partitioning the first strand and the second strand into a plurality of droplets, wherein each droplet contains only the first strand or only the second strand;

detecting a presence of the first strand or the second strand in each droplet; and

quantifying a number of droplets in which the first strand or the second strand is present, wherein quantifying comprises counting droplets.

2. The method of claim 1 wherein detecting comprises conducting a PCR reaction in each droplet, thereby changing a fluorescent property of each droplet, and assessing the fluorescent property of each droplet.

3. The method of claim 2, wherein conducting a PCR reaction comprises annealing a first strand and a second strand independently with a primer.

4. The method of claim 3, wherein the primer anneals with a human sequence mutation.

5. The method of claim 3, wherein the primer anneals with a pathogenic sequence.

6. The method of claim 3, wherein the primer anneals with a genetic marker for a disease.

7. The method of claim 1, wherein denaturing comprises heating the double-stranded DNA.

8. The method of claim 7, wherein heating comprises raising the temperature of the double-stranded DNA to at least 90°

C.

9. The method of claim 7, further comprising rapidly cooling the first strand and the second strand after heating the double-stranded DNA.

10. The method of claim 9, wherein rapidly cooling comprises reducing the temperature of the first or second strand to less than 20°

C. in less than 60 seconds.

11. The method of claim 1, wherein each droplet is substantially surrounded by an immiscible carrier fluid in a microfluidic device.

12. The method of claim 1, further comprising detecting an absence of the first strand or the second strand in each droplet.

13. The method of claim 1, further comprising determining an amount of DNA in the sample based on the quantifying step.

14. A method for determining the progression of a disease in a subject, the method comprising:

obtaining a biological sample comprising a plurality of double-stranded DNAs from a subject;

dissociating the plurality of double-stranded DNAs into a plurality of first strands and a plurality of second strands;

partitioning the plurality of first strands and the plurality of second strands into a plurality of partitioned portions, at least some of the partitioned portions having only a first strand or only a second strand;

detecting the presence or absence of a targeted sequence in the plurality of partitioned portions; and

determining the number of occurrences of the targeted sequence by counting droplets comprising either the first strand or the second strand, wherein the number of occurrences of the targeted sequence is indicative of a progression of a disease.