Methods for beaming
Improvements on the basic method used for BEAMing increase sensitivity and increase the signal-to-noise ratio. The improvements have permitted the determination of intrinsic error rates of various DNA polymerases and have permitted the detection of rare and subtle mutations in DNA isolated from plasma of cancer patients.
1. A method for detecting rare DNA mutations in a sample, comprising:
performing real-time PCR on a sample using a high fidelity DNA polymerase to form a set of first amplicons;
forming a water-in-oil microemulsion, comprising an aqueous phase and an oil phase, wherein the microemulsion comprises the set of first amplicons and beads, wherein the beads are bound to a plurality of molecules of a primer for amplifying the set of first amplicons;
amplifying the set of first amplicons in the water-in-oil microemulsion, whereby product beads are formed which are bound to a set of second amplicons;
separating the aqueous phase from the oil phase of the microemulsion;
breaking the microemulsion;
associating a nucleotide of a wild-type gene from the set of second amplicons with a fluorescently-labelled tag and associating at least one nucleotide of a genetic mutation from the set of second amplicons with a different fluorescently-labelled tag; and
analyzing single beads to the exclusion of bead doublets and bead aggregates, wherein single beads are analyzed by flow cytometry to measure the signal associated with individual beads, thereby detecting rare DNA mutations in a sample.
2. The method of claim 1 , wherein the sample is obtained from blood, urine, or stool.
3. The method of claim 1 , wherein the sample is obtained from plasma.
4. The method of claim 1 , the microemulsion are formed with a tissue homogenizer.
5. The method of claim 1 , wherein the microemulsion are formed with a mechanical tissue homogenizer.
6. The method of claim 1 , wherein the microemulsion are formed with a rotor-stator tissue homogenizer.
7. The method of claim 1 , wherein two high fidelity polymerases are used in parallel and compared to ascertain relative fidelity.
8. The method of claim 1 , wherein the microemulsion comprise a thermostable emulsifying agent.
9. The method of claim 1 , wherein the associating step comprises single base extension.
10. The method of claim 1 , wherein the associating step comprises sequencing by synthesis.
11. The method of claim 1 , wherein the method detects at least 0.2% mutant DNA fragments within the sample.