Methods for beaming

Patent metadata

Field	Value
Publication number	US-9360526-B2
Application number	US-9139506-A
Country	US
Kind code	B2
Filing date	Oct 20, 2006
Priority date	Oct 24, 2005
Publication date	Jun 7, 2016
Grant date	Jun 7, 2016

Abstract

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.

First claim

We claim:

1. A method for amplifying a region of analyte DNA molecules, comprising:
amplifying a region of analyte DNA molecules using a high fidelity DNA polymerase to form a set of first amplicons;
forming microemulsions comprising said first amplicons and reagent beads, wherein the reagent beads are bound to a plurality of molecules of a primer for amplifying the set of first amplicons;
amplifying the first amplicons in the microemulsions, whereby product beads are formed which are bound to a plurality of copies of second amplicons;
breaking the microemulsions;
amplifying the second amplicons bound to the product beads using rolling circle amplification to form third amplicons, wherein the rolling circle amplification employs a circularizable probe, said probe comprising a first and a second region of complementarity with a third and a fourth region on said second amplicons, wherein said first and second regions are non-contiguous on said probe, and wherein said third and fourth regions are non-contiguous on said second amplicons, wherein said second amplicons comprise a fifth region of 1-30 nucleotides between said third and fourth regions, wherein upon hybridization of the circularizable probe to the second amplicons a DNA polymerase fills in 1-30 nucleotides complementary to the fifth region, between said first and second region of complementarity of the circularizable probe, and template-driven ligation of the ends of the circularizable probe forms a circle; and
analyzing single beads to the exclusion of bead doublets and bead aggregates.

2. The method of claim 1 wherein the microemulsions comprise a thermostable emulsifying agent.

3. The method of claim 1 wherein the high fidelity polymerase has an error rate of less than 10 −5 errors per basepair per cycle.

4. The method of claim 1 wherein the high fidelity polymerase has an error rate of less than 5×10 −6 errors per basepair per cycle.

5. The method of claim 1 wherein the high fidelity polymerase has an error rate of less than 10 −6 errors per basepair per cycle.

6. The method of claim 1 wherein the first amplicons are less than or equal to 300 bp.

7. The method of claim 1 wherein the first amplicons are less than or equal to 200 bp.

8. The method of claim 1 wherein the first amplicons are less than or equal to 100 bp.

9. The method of claim 1 wherein the analyte DNA molecules are obtained from plasma.

10. The method of claim 1 wherein the microemulsions are formed with a tissue homogenizer.

11. The method of claim 1 wherein the microemulsions are formed with a rotor-stator tissue homogenizer.

12. The method of claim 1 further comprising the step of:
determining a sequence feature of the third amplicons by single base extension with at least two differentially labeled dideoxyribonucleotides of a primer bound to said third amplicons.

13. The method of claim 12 wherein the labeled dideoxyribonucleotides are fluorescent and flow cytometry is used to detect the labeled dideoxyribonucleotides present on the product beads.

14. The method of claim 12 further comprising the step of discarding from analysis beads which display two or more differentially labeled dideoxyribonucleotides extended onto primers bound to second amplicons bound to the beads.

15. The method of claim 12 wherein prior to the step of determining, the product beads are subjected to denaturing conditions whereby the third amplicons are separated into single strands, and wherein single strands which are not bound to the product beads are discarded.

16. The method of claim 12 wherein prior to the step of determining, the product beads are incubated with unlabeled dideoxynucleotides.

17. The method of claim 1 wherein two high-fidelity polymerases are used in parallel and compared to ascertain relative fidelity.

18. The method of claim 1 wherein the analyte DNA has been treated with a potential mutagen.

19. The method of claim 1 wherein the analyte DNA is obtained from blood, urine, or stool of a cancer patient.

20. The method of claim 1 wherein the analyte DNA is obtained from plasma of a pregnant woman.

21. The method of claim 1 wherein the third amplicons are used as templates for nucleotide sequencing reactions.

22. The method of claim 1 wherein the rolling circle amplification produces an amount of third amplicon which produces a signal that is from about 1500 to about 9000-fold the signal produced by the second amplicon.

23. The method of claim 1 wherein the rolling circle amplification produces an amount of third amplicon which produces a signal that is from about 1500 to about 7000-fold the signal produced by the second amplicon.

24. The method of claim 1 wherein the rolling circle amplification produces an amount of third amplicon which produces a signal that is from about 1500 to about 2200 fold the signal produced by the second amplicon.

25. The method of claim 1 wherein the rolling circle amplification produces an amount of third amplicon which produces a signal that is from about 7000 to about 9000 fold the signal produced by the second amplicon.

26. The method of claim 1 wherein the rolling circle amplification is performed for 5 minutes to 15 minutes.

27. The method of claim 1 wherein the beads are analyzed by flow cytometry.

Assignees

• Vogelstein Bert
• Diehl Frank
• Kinzler Kenneth W
• Li Meng
• Univ Johns Hopkins

Inventors

• Vogelstein Bert
• Diehl Frank
• Kinzler Kenneth W
• Li Meng

Classifications

• A61P9/10

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• G01R31/52

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• H02H1/0015

  Using arc detectors · CPC title

• H02H3/33

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• G01R31/3277 Primary

  of low voltage devices, e.g. domestic or industrial devices, such as motor protections, relays, rotation switches · CPC title