Analysis of nucleic acids

• US 10,167,509 B2
• Filed: 09/08/2015
• Issued: 01/01/2019
• Est. Priority Date: 02/09/2011
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Regan, John F., Saxonov, Serge, Lucero, Michael Y., Hindson, Benjamin J., Belgrader, Phillip, Dube, Simant, So, Austin P., Mellen, Jeffrey C., Heredia, Nicholas J., Ness, Kevin D., Colston, Jr., Billy W.
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US14/848,311
• Publication Number:
  US 20160076099A1
• Time in Patent Office: 1,211 Days
• 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 identifying a plurality of target nucleic acids as being present on the same polynucleotide, the method comprising:

a) separating a sample comprising a plurality of polynucleotides into a first subsample and a second subsample, wherein the polynucleotides comprise a first target nucleic acid and a second target nucleic acid;

b) contacting only the first subsample of the first and second subsamples with an agent capable of physically separating the first target nucleic acid from the second target nucleic acid if both target nucleic acids are present on the same polynucleotide;

c) following step b, separating the first subsample into a first set of partitions;

d) determining a first number of partitions in the first set of partitions that comprise at least one of the target nucleic acids, or a first number of partitions in the first set of partitions that comprise both of the target nucleic acids;

e) separating the second subsample into a second set of partitions;

f) determining a second number of partitions in the second set of partitions that comprise at least one of the target nucleic acids, or a second number of partitions in the second set of partitions that comprise both of the target nucleic acids; and

g) comparing a first value corresponding to the first number obtained in step d with a second value corresponding to the second number obtained in step f to determine whether the first target nucleic acid and the second target nucleic acid are present within the same polynucleotide;

wherein the first and second numbers both represent partitions that comprise at least one of the target nucleic acids or both represent partitions that comprise both of the target nucleic acids.

2. The method of claim 1, wherein the first and second target nucleic acids comprise the same sequence.

3. The method of claim 1, wherein the agent includes a restriction enzyme.

4. The method of claim 3, wherein the restriction enzyme recognizes a site between the first and second target nucleic acids.

5. The method of claim 1, wherein linkage of the first and second target nucleic acids is indicated when the number obtained in step d is significantly lower than the number obtained in step f.

6. The method of claim 1, wherein the first and second target nucleic acids are located within 1 megabase of each other.

7. The method of claim 1, wherein the first and second target nucleic acids are not physically separated in the second subsample.

8. The method of claim 1, further comprising determining whether the first and second target nucleic acids are on the same chromosome or different chromosomes.

9. The method of claim 1, wherein the sequence of the first target nucleic acid is different from the sequence of the second target nucleic acid, wherein the first number represents partitions of the first set that comprise both of the target nucleic acids, and wherein the second number represents partitions of the second set that comprise both of the target nucleic acids.

10. The method of claim 9, wherein the sequence of the first target nucleic acid is a genetic variation of the second target nucleic acid.

11. The method of claim 10, wherein the genetic variation is a single nucleotide polymorphism.

12. The method of claim 9, wherein the first and second target nucleic acids are within the same gene.

13. The method of claim 1, wherein the first number represents partitions of the first set that comprise at least one of the target nucleic acids, and wherein the second number represents partitions of the second set that comprise at least one of the target nucleic acids.

14. The method of claim 13, wherein linkage of the first and second target nucleic acids is indicated when the number obtained in step d is significantly higher than the number obtained in step f.

15. The method of claim 1, wherein the first value is an estimated copy number of a sequence in the first subsample, and wherein the second value is an estimated copy number of the sequence in the second subsample.

16. The method of claim 1, further comprising a step of amplifying the first target nucleic acid and the second target nucleic acid in each of the first and second sets of partitions using the same pair of primers.

17. The method of claim 16, wherein each of the first set of partitions and the second set of partitions comprises the same labeled probe configured to report amplification of the first target nucleic acid and amplification of the second nucleic acid.

18. The method of claim 1, further comprising a step of detecting fluorescence from each of the first and second sets of partitions, wherein steps d and f, respectively, are based on fluorescence detected from the first set of partitions and the second set of partitions.