Compositions and methods for clonal amplification and analysis of polynucleotides

• US 20060269934A1
• Filed: 03/16/2006
• Published: 11/30/2006
• Est. Priority Date: 03/16/2005
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Nordman, Eric S., Woudenberg, Timothy M., Heiner, Cheryl
• Application Number:
  US11/377,763
• Publication Number:
  US 20060269934A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

Compositions and methods of use are disclosed for clonally amplifying and analyzing one or more polynucleotides.

Claims

1. A method of analyzing a plurality of polynucleotides comprising:

a) amplifying a plurality of polynucleotides under conditions suitable to produce a plurality of multiplex amplicons;

b) clonally amplifying said multiplex amplicons to produce a plurality of clonal amplicons; and

c) analyzing said plurality of clonal amplicons.

2. The method according to claim 1, wherein said plurality of polynucleotides is at least about 100 polynucleotides.

3. The method according to claim 1, wherein said plurality of polynucleotides is at least about 1000 polynucleotides.

4. The method according to claim 1, wherein said plurality of polynucleotides is at least about 10,000 polynucleotides.

5. The method according to claim 1, wherein said plurality of polynucleotides is at least about 100,000 polynucleotides.

6. The method according to claim 1, wherein said plurality of polynucleotides is at least about 1,000,000 polynucleotides.

9. The method according to claim 1, wherein said multiplex amplicons are clonally amplified in hydrophilic compartments of an inverse emulsion.

10. The method according to claim 9, wherein said hydrophilic compartments are disposed upon a surface.

11. The method according to claim 10, wherein said surface comprises primers suitable for clonally amplifying said multiplex amplicons.

12. The method according to claim 11, wherein said primers are hybridized to said multiplex amplicons.

13. The method according to claim 12, wherein said clonal amplicons are attached to said surface.

14. The method according to claim 1, wherein said conditions suitable for producing said plurality of multiplex amplicons comprise multiple rounds of a thermocycling reaction comprising forward and reverse amplification primer pairs, a thermostable polymerase, and deoxynucleotide triphosphate suitable for DNA synthesis.

15. The method according to claim 14, wherein said multiple rounds of a thermocycling reaction terminates before said reaction reaches a plateau.

16. The method according to claim 14, wherein said forward primers comprises a forward universal sequence and said reverse primers comprise a reverse universal sequence.

22. The method according to claim 1, wherein said analyzing comprises sequencing said plurality of clonal amplicons.

23. The method according to claim 22, wherein said sequencing comprising sequencing in parallel.

24. The method according to claim 22, wherein said sequencing is massively parallel signature sequence.

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28. A method of amplifying a plurality of polynucleotides comprising:

hybridizing polynucleotides isolated in hydrophilic compartments of an inverse emulsion to primers, wherein said primers are attached to a surface and said compartments are disposed upon said surface, and wherein said polynucleotides and said primers are hybridized in the presence of a polymerase and nucleotide triphosphates suitable for nucleic acid synthesis and under conditions suitable for said polymerase to extend said primers, whereby said polynucleotides are amplified.

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35. A method of analyzing a plurality of polynucleotides comprising:

a) clonally amplifying a plurality of polynucleotides in hydrophilic compartments of an inverse emulsion in the presence of primers, a polymerase, and nucleotide triphosphates suitable for nucleic acid synthesis, wherein said primer is attached to a surface and said compartment is disposed upon said surface, and under conditions suitable to produce a plurality of clonal amplicons attached to said surface; and

b) analyzing said plurality of clonal amplicons.

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51. A composition comprising an inverse emulsion and an oligonucleotide attached to a surface, wherein said inverse emulsion comprises at least one hydrophilic compartment that is disposed upon a said surface and wherein said oligonucleotide is in contact with said hydrophilic compartment.

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