Method and compositions for detection and enumeration of genetic variations

Patent metadata

Field	Value
Publication number	US-9328343-B2
Application number	US-201113311120-A
Country	US
Kind code	B2
Filing date	Dec 5, 2011
Priority date	Jul 5, 2003
Publication date	May 3, 2016
Grant date	May 3, 2016

Abstract

Many areas of biomedical research depend on the analysis of uncommon variations in individual genes or transcripts. Here we describe a method that can quantify such variation at a scale and ease heretofore unattainable. Each DNA molecule in a collection of such molecules is converted into a single particle to which thousands of copies of DNA identical in sequence to the original are bound. This population of beads then corresponds to a one-to-one representation of the starting DNA molecules. Variation within the original population of DNA molecules can then be simply assessed by counting fluorescently-labeled particles via flow cytometry. Millions of individual DNA molecules can be assessed in this fashion with standard laboratory equipment. Moreover, specific variants can be isolated by flow sorting and employed for further experimentation. This approach can be used for the identification and quantification of rare mutations as well as to study variations in gene sequences or transcripts in specific populations or tissues.

First claim

We claim:

1. A method for analyzing nucleic acid sequences comprising:
(a) delivering a plurality of molecules of a fragment of deoxyribonucleic acid into aqueous microreactors in a water-in-oil emulsion such that a plurality of aqueous microreactors comprise a single molecule of the fragment of deoxyribonucleic acid, a single bead capable of hybridizing to but not hybridized to the fragment of deoxyribonucleic acid, and reagents necessary to perform deoxyribonucleic acid amplification;
(b) amplifying the fragment of deoxyribonucleic acid in the microreactors to form amplified copies of the fragment of deoxyribonucleic acid bound to beads in the microreactors; and
(c) determining presence of amplified copies of a strand of the fragment of deoxyribonucleic acid bound to a bead.

2. The method of claim 1 wherein step (b) is accomplished using polymerase chain reaction.

3. The method of claim 1 wherein the step of amplifying converts less than 10% of the beads present in the microreactors into beads bound to amplified copies of the fragment of deoxyribonucleic acid.

4. The method of claim 1 wherein the step of amplifying employs copies of a primer which are bound to the beads and copies of the primer which are not bound to the beads.

5. The method of claim 1 further comprising the step of:
generating the plurality of molecules of a fragment of deoxyribonucleic acid, wherein said step of generating is performed prior to the step of delivering.

6. The method of claim 1 further comprising the step of:
amplifying by polymerase chain reaction a nucleic acid fragment to form the plurality of molecules of a fragment of deoxyribonucleic acid, wherein said step of amplifying is performed prior to the step of delivering.

7. The method of claim 1 wherein the step of determining presence is performed using flow cytometry.

8. A method for analyzing nucleotide sequences from a sample selected from the group consisting of urine, blood, sputum, stool, tissue, and saliva of a eukaryotic organism, comprising:
forming microemulsions comprising one or more species of analyte DNA molecules, such that a plurality of aqueous compartments comprise a single species of the analyte DNA which is not hybridized to a primer bound to a reagent bead; and then
amplifying analyte DNA molecules in the microemulsions in the presence of reagent beads, wherein the reagent beads are bound to a plurality of molecules of a primer for amplifying the analyte DNA molecules, whereby product beads are formed which are bound to a plurality of copies of the single species of analyte DNA molecule;
separating the product beads from analyte DNA molecules which are not bound to product beads; and
determining presence of the single species of analyte DNA molecule which is bound to the product beads.

9. The method of claim 8 wherein prior to the step of separating, the microemulsions are broken by addition of one or more detergents.

10. The method of claim 8 wherein the step of amplifying employs additional copies of the primer which are not bound to the reagent bead.

11. The method of claim 8 wherein the analyte DNA molecules are genomic DNA.

12. The method of claim 8 wherein the analyte DNA molecules are PCR products made from genomic DNA.

13. The method of claim 8 wherein the analyte DNA molecules are derived from a single individual.

14. The method of claim 8 wherein the reagent beads are magnetic.

15. The method of claim 8 wherein the step of amplifying converts less than 10% of the reagent beads present in the microemulsions into product beads.

16. The method of claim 8 further comprising the step of:
amplifying by polymerase chain reaction one or more species of analyte DNA molecules to form the one or more species of analyte DNA molecules, wherein said step of amplifying is performed prior to the step of forming microemulsions.

17. The method of claim 8 wherein the step of determining presence is performed using flow cytometry.

18. The method of claim 8 further comprising the step of obtaining DNA from the sample selected from the group consisting of urine, blood, sputum, stool, tissue, and saliva of a eukaryotic organism, prior to forming microemulsions.

19. A method for analyzing nucleotide sequences, comprising:
forming microemulsions comprising one or more species of analyte DNA molecules which is not hybridized to a primer bound to a reagent bead; and then
amplifying the analyte DNA molecules in the microemulsions in the presence of reagent beads, wherein the reagent beads are bound to a plurality of molecules of a primer for amplifying the analyte DNA molecules, whereby product beads are formed which are bound to a plurality of copies of one of the one or more species of analyte DNA molecules, wherein the step of amplifying converts less than 10% of the reagent beads present in the microemulsions into product beads;
separating the product beads from analyte DNA molecules which are not bound to product beads; and
determining presence of a strand of the one species of analyte DNA molecule which is bound to the product beads.

20. The method of claim 19 wherein the one or more species of analyte DNA molecules are a single species of analyte DNA molecules.

21. The method of claim 19 wherein the step of amplifying employs additional copies of the primer which are not bound to the reagent bead.

22. The method of claim 19 further comprising the step of:
amplifying by polymerase chain reaction one or more species of analyte DNA molecules to form the one or more species of analyte DNA molecules, wherein said step of amplifying is performed prior to the step of forming microemulsions.

23. The method of claim 19 wherein the step of determining presence is performed using flow cytometry.

Assignees

• Dressman Devin
• Yan Hai
• Kinzler Kenneth W
• Vogelstein Bert
• Univ Johns Hopkins

Inventors

• Dressman Devin
• Yan Hai
• Kinzler Kenneth W
• Vogelstein Bert

Classifications

• C12Q1/6858 Primary

  Allele-specific amplification · CPC title

• C12N15/1075 Primary

  by coupling phenotype to genotype, not provided for in other groups of this subclass · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C07H21/04

  with deoxyribosyl as saccharide radical · CPC title

• C12Q2565/537

  characterised by the capture oligonucleotide acting as a primer · CPC title