Method and compositions for detection and enumeration of genetic variations

Patent metadata

Field	Value
Publication number	US-10604797-B2
Application number	US-201615096355-A
Country	US
Kind code	B2
Filing date	Apr 12, 2016
Priority date	Jul 5, 2003
Publication date	Mar 31, 2020
Grant date	Mar 31, 2020

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 composition comprising a plurality of beads, wherein the beads are magnetic, wherein each of said plurality of beads comprises a plurality of bound polynucleotides, wherein the polynucleotides in the composition are heterogeneous with respect to the presence or absence of a mutation, and wherein on at least 1% of said beads the plurality of bound polynucleotides is homogeneous, and wherein the composition comprises an emulsion forming aqueous compartments, wherein at least a portion of the aqueous compartments comprise a bead.

2. The composition of claim 1 wherein on at least 5% of said beads the plurality of bound polynucleotides is homogeneous.

3. The composition of claim 1 wherein on at least 10% of said beads the plurality of bound polynucleotides is homogeneous.

4. The composition of claim 1 wherein on at least 50% of said beads the plurality of bound polynucleotides is homogeneous.

5. The composition of claim 1 wherein the plurality of bound polynucleotides is greater than 100.

6. The composition of claim 1 which is a liquid.

7. The composition of claim 1 which comprises agarose.

8. The composition of claim 1 wherein the polynucleotides in the composition differ in the presence or absence of an insertion mutation.

9. The composition of claim 1 wherein the bound polynucleotides were made by amplification of a template in a test sample, wherein the beads on which the plurality of bound polynucleotides is homogeneous comprise at least a first and second species of polynucleotide, wherein the beads comprising the first species of polynucleotide and the beads comprising the second species of polynucleotide are present in the composition in the same ratio as the first and second species of polynucleotide were present in the test sample.

Assignees

• Univ Johns Hopkins

Inventors

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

Classifications

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C07H21/04

  with deoxyribosyl as saccharide radical · CPC title

• C12Q1/6858 Primary

  Allele-specific amplification · CPC title

• C12N15/1075

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

• C12Q2563/149

  Particles, e.g. beads · CPC title