Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations

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 magnetic 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. This approach is called BEAMing on the basis of four of its principal components (beads, emulsion, amplification, and magnetics). 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 used for further experimentation. BEAMing 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.

Detection and quantification of mutations in the plasma of patients with colorectal tumors

“…This oligonucleotide was coupled to streptavidin-coated magnetic beads (MyOne, Dynal, Oslo) according to the protocol described in ref. 16. The water-in-oil emulsions were prepared by modifications of the methods described by Ghadessy and Holliger (17) 5 per l oligonucleotide-coupled beads, and 0.1 pg͞ l template DNA.…”

Sequence and structural variation in a human genome uncovered by short-read, massively parallel ligation sequencing using two-base encoding

“…Emulsion PCR was performed according to Dressman et al (2003) with a few minor modifications (Supplement Emulsion Methods). Since limited dilution of DNA is utilized to produce clonal bead amplification, 70%-80% of the beads in any given emulsion are unamplified beads.…”

Analysis of Mutations in DNA Isolated From Plasma and Stool of Colorectal Cancer Patients

“…BEAMing was named after its components-beads, emulsions, amplification, and magnetics-and essentially converts single DNA template molecules to single beads containing tens of thousands of exact copies of the template. 6 We used this method to determine how frequently mutations could be detected in the DNA from plasma or stool of the same patients as well as to investigate other parameters that could be useful in designing clinically applicable DNA-based tests in the future.…”