Method and Compositions for Detection and Enumeration of Genetic Variations

• US 20120183967A1
• Filed: 12/05/2011
• Published: 07/19/2012
• Est. Priority Date: 07/05/2003
• Status: Active Grant

• Current Assignee: Johns Hopkins University
• Original Assignee: Johns Hopkins University
• Inventors: KINZLER, Kenneth W., VOGELSTEIN, Bert, DRESSMAN, Devin, YAN, Hai
• Granted Patent: US 9,328,343 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

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.

Claims

1. A method for analyzing nucleic acid sequences comprising:

(a) generating a plurality of molecules of a double-stranded fragment of deoxyribonucleic acid;

(b) delivering the plurality of molecules of the double-stranded 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 double-stranded deoxyribonucleic acid, a single bead capable of hybridizing to one strand of the double-stranded fragment of deoxyribonucleic acid, and reagents necessary to perform deoxyribonucleic acid amplification;

(c) amplifying the fragment of deoxyribonucleic acid in the microreactors to form amplified copies of the double-stranded fragment of deoxyribonucleic acid bound to beads in the microreactors;

(d) determining presence of amplified copies of a strand of the double-stranded fragment of deoxyribonucleic acid bound to a bead.

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

14. The method of claim 1 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 1 wherein the step of amplifying employs additional copies of the primer which are not bound to the reagent bead.

3. 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 double-stranded analyte DNA molecules, such that a plurality of aqueous compartments comprise a single species of the double-stranded analyte DNA;

amplifying double-stranded 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 double-stranded analyte DNA molecules, whereby product beads are formed which are bound to a plurality of copies of one strand of the single species of analyte DNA molecule;

separating the product beads from double-stranded analyte DNA molecules which are not bound to product beads;

determining presence of a strand of the single species of double-stranded analyte DNA molecule which is bound to the product beads.

5. The method of claim 3 wherein prior to the step of separating, the microemulsions are broken by addition of one or more detergents.

6. The method of claim 3 wherein the step of amplifying employs additional copies of the primer which are not bound to the reagent bead.

7. The method of claim 3 wherein the double-stranded analyte DNA molecules are genomic DNA.

8. The method of claim 3 wherein the double-stranded analyte DNA molecules are PCR products made from genomic DNA.

9. The method of claim 3 wherein the double-stranded analyte DNA molecules are derived from a single individual.

10. The method of claim 3 wherein the reagent beads are magnetic.

11. The method of claim 3 wherein the single species of double-stranded analyte DNA molecules are double-stranded DNA fragments.

15. The method of claim 3 wherein the step of amplifying converts less than 10% of the reagent beads present in the microemulsions into product beads.

4. A method for analyzing nucleotide sequences, comprising:

forming microemulsions comprising one or more species of double-stranded analyte DNA molecules;

amplifying the double-stranded 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 double-stranded analyte DNA molecules, whereby product beads are formed which are bound to a plurality of copies of one strand of the one species of analyte DNA molecule, 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 double-stranded analyte DNA molecules which are not bound to product beads;

determining presence of a strand of the one species of analyte DNA molecule which is bound to the product beads.

12. The method of claim 4 wherein the one or more species of double-stranded analyte DNA molecules are a single species of double-stranded analyte DNA molecules.

13. The method of claim 4 wherein the one or more species of double-stranded analyte DNA molecules are double-stranded DNA fragments.

17. The method of claim 4 wherein the step of amplifying employs additional copies of the primer which are not bound to the reagent bead.