Methods for beaming

Patent metadata

Field	Value
Publication number	US-10150991-B2
Application number	US-201615174427-A
Country	US
Kind code	B2
Filing date	Jun 6, 2016
Priority date	Oct 24, 2005
Publication date	Dec 11, 2018
Grant date	Dec 11, 2018

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 analyzing nucleotide sequence variations, 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 using rolling circle amplification to form third amplicons;
determining a sequence feature of third amplicons by single base extension of a primer bound to said second amplicons using at least two differentially labeled dideoxyribonucleotides;
wherein the high fidelity polymerase has an error rate of less than 10 −5 errors per basepair per cycle.

2. 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.

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

4. The method of claim 1 , wherein the first amplicon is less than or equal to 300 bp.

5. The method of claim 1 , wherein the first amplicon is less than or equal to 200 bp.

6. The method of claim 1 , wherein the first amplicon is less than or equal to 100 bp.

7. The method of claim 1 , wherein the at least two labeled dideoxyribonucleotides are fluorescent and flow cytometry is used to detect the labeled dideoxyribonucleotides present on the product beads.

8. The method of claim 1 , further comprising the step of discarding from analysis beads which display two or more differentially labeled dideoxyribonucleotides extended onto primers bound to third amplicons bound to the beads.

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

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 mechanical tissue homogenizer.

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

13. The method of claim 1 wherein prior to the step of determining, the product beads are subjected to denaturing conditions whereby the second amplicons are separated into single strands, and wherein single strands which are not bound to the product beads are discarded.

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

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

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

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

18. The method of claim 1 , wherein prior to the step of determining, the product beads are incubated with unlabeled deoxynucleotides.

19. 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 using rolling circle amplification to form third amplicons.

20. The method of claim 19 , 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, template-driven ligation of the ends of the circularizable probe forms a circle.

21. The method of claim 19 , wherein the rolling circle amplification employs a padlock 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 contiguous on said probe, and wherein said third and fourth regions are contiguous on said second amplicons, wherein upon hybridization of the padlock probe to the second amplicons, template-driven ligation of the ends of the padlock probe forms a circle.

22. The method of claim 21 , wherein the analyte DNA has been treated with a potential mutagen.

23. The method of claim 21 , 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.

24. The method of claim 21 , wherein prior to the step of determining, the product beads are incubated with unlabeled deoxynucleotides.

25. The method of claim 19 , 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.

26. The method of claim 19 , wherein two high-fidelity polymerases are used in parallel and compared to ascertain relative fidelity.

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

28. The method of claim 19 , wherein the analyte DNA is obtained from a plasma of a pregnant woman.

29. The method of claim 19 , wherein the third amplicons are used as templates for nucleotide sequencing reactions.

Assignees

• 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

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• H02H3/33

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• G01R31/3277

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