Analysis of nucleic acids

• US 9,127,312 B2
• Filed: 02/09/2012
• Issued: 09/08/2015
• Est. Priority Date: 02/09/2011
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Regan, John Frederick, Saxonov, Serge, Lucero, Mike, Hindson, Ben, Belgrader, Phil, Dube, Simant, So, Austin, Mellen, Jeffrey Clark, Heredia, Nicholas Jack, Ness, Kevin, Colston, Bill
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US13/385,277
• Publication Number:
  US 20120322058A1
• Time in Patent Office: 1,307 Days
• Field of Search: 435/6.1, 435/91.2, 536/22.1, 536/23.1, 536/24.3
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2537/16 Assays for determining copy number or wherein the copy number is of special importance
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• 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 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/172 Haplotypes

Abstract

Provided herein are improved methods, compositions, and kits for analysis of nucleic acids. The improved methods, compositions, and kits can enable copy number estimation of a nucleic acid in a sample. Also provided herein are methods, compositions, and kits for determining the linkage of two or more copies of a target nucleic acid in a sample (e.g., whether the two or more copies are on the same chromosome or different chromosomes) or for phasing alleles.

Claims

1. A method of detecting variations in copy number of a target nucleic acid, the method comprising:

a. contacting a sample comprising a plurality of polynucleotides with at least one agent, wherein each of one or more of polynucleotides comprises multiple copies of a target nucleic acid;

b. subjecting the sample to conditions that enable the agent to cleave a specifically-selected target site between the multiple copies of the target nucleic acid, thereby separating the multiple copies from one another;

c. separating the sample contacted with the agent into a plurality of droplets;

d. subjecting the target nucleic acid to an amplification reaction in droplets;

e. detecting amplification data for the target nucleic acid from individual droplets;

f. enumerating a number of droplets comprising the target nucleic acid based on the amplification data; and

g. determining a copy number of the target nucleic acid based on the step of enumerating.

2. The method of claim 1, wherein the target nucleic acid is are present at an average concentration of less than about five copies per droplet.

3. The method of claim 1, wherein the multiple copies have identical sequences.

4. The method of claim 1, wherein the specifically-selected target site is a site capable of digestion with a restriction enzyme.

5. The method of claim 1, wherein the agent is one or more restriction enzymes.

6. The method of claim 1, wherein the specifically-selected site and the target nucleic acid are located within the same gene.

7. The method of claim 1, wherein the target nucleic acid is correlated with a disease or disorder.

8. The method of claim 1, further comprising enumerating a number of droplets comprising a reference nucleic acid.

9. The method of claim 8, wherein the reference nucleic acid is present at a fixed number of copies in a genome from which the sample is derived.

10. The method of claim 8, wherein the reference nucleic acid is a housekeeping gene.

11. The method of claim 8, wherein the reference nucleic acid is present at two copies per diploid genome from which the sample is derived.

12. The method of claim 8, wherein determining the copy number of the target nucleic acid comprises dividing the number of droplets comprising the target nucleic acid by the number of droplets comprising the reference nucleic acid.

13. The method of claim 1, wherein the agent does not cut the sequence of the target nucleic acid.

14. The method of claim 8, wherein the agent does not cut the sequence of the reference nucleic acid.

15. The method of claim 5, wherein the one or more restriction enzymes have more than one recognition sequence between the multiple copies of the target nucleic acid.

16. The method of claim 5, wherein the one or more restriction enzymes do not exhibit significant star activity.

17. The method of claim 5, wherein the one or more restriction enzymes comprise two or more restriction enzymes.

18. The method of claim 5, wherein software is used to select the one or more restriction enzymes.

19. The method of claim 5, wherein the one or more restriction enzymes are heat-inactivated after digesting the polynucleotides.

20. The method of claim 19, wherein a temperature of the heat inactivation is below a melting point of the restricted target fragments in order to maintain the double-stranded nature of the target fragments.

21. The method of claim 5, wherein a control restriction enzyme digest is performed to measure an efficiency of digestion of nucleic acid by the one or more restriction enzymes.

22. The method of claim 1, wherein a percentage of linked target sequences that are fragmented in the sample is determined.

23. The method of claim 1, wherein less than 1 megabase separates the multiple copies of the target nucleic acid.

24. The method of claim 1, wherein less than 1 kilobase separates the multiple copies of the target nucleic acid.

25. The method of claim 1, wherein subjecting the target nucleic acid to an amplification reaction includes conducting a PCR reaction.

26. The method of claim 1, wherein the method does not comprise a sequencing reaction.

27. A method of detecting variations in copy number of a target nucleic acid comprising:

a. obtaining a sample comprising (i) a plurality of polynucleotides, wherein at least one of the polynucleotides comprises multiple copies of a target nucleic acid;

(ii) a probe with a fluorescent label to detect the target nucleic acid; and

(iii) reagents to enable a PCR reaction;

b. separating the sample into a plurality of spatially-isolated partitions, wherein the spatially-isolated partitions comprise on average less than five copies of the target nucleic acid;

c. subjecting the sample to a PCR amplification reaction in order to detect the target nucleic acid;

d. detecting the fluorescence intensity of the fluorescent label before any of the reagents for the PCR reaction become limiting;

e. enumerating the number of partitions that have a fluorescence intensity above a threshold value indicative of one copy of the target nucleic acid;

f. enumerating the number of partitions that have a fluorescence intensity above a threshold value indicative of multiple copies of the target nucleic acid; and

g. calculating the copy number of the target nucleic acid based on the numbers obtained in step e and step f.

28. The method of claim 27, wherein increased copy number of the target nucleic acid is correlated with a disease or disorder.