Clonal pre-amplification in emulsion

• US 9,650,629 B2
• Filed: 06/27/2011
• Issued: 05/16/2017
• Est. Priority Date: 07/07/2010
• Status: Active Grant

• Current Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Original Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Inventors: Heckel, Tobias, Froehlich, Thomas
• Primary Examiner(s): BERTAGNA, ANGELA MARIE
• Application Number:
  US13/169,110
• Publication Number:
  US 20120010086A1
• Time in Patent Office: 2,150 Days
• Field of Search: None
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR

Abstract

Disclosed is a process for clonal pre-amplification of a nucleic acid involving the steps of (i) providing a plurality of different nucleic acid molecules (b) attaching adaptor sequences to the 3′ ends and 5′ ends of the nucleic acid molecules (c) preparing a water in oil emulsion wherein the majority of water droplets comprises one or none member of the plurality of different nucleic acid molecules (d) clonally amplifying the plurality of different nucleic acid molecules. In particular, the different nucleic acid molecules are mRNA molecules.

Claims

1. A method of generating an unbiased population of amplified cDNA comprising the steps of:

(a) providing a plurality of different single-stranded RNA molecules, the plurality comprising different single-stranded RNA molecules present at different representation frequencies in the plurality,

(b)-(b1) hybridizing a first adaptor molecule to the 3′

ends of RNA molecules of said plurality,

the first adaptor molecule comprising a 5′

terminal part comprising a primer binding site, and

a 3′

terminal part comprising an oligo (dT) sequence at least 5 nucleotides in length, a randomized sequence at least 5 nucleotides in length, or a specific sequence for a gene or gene family,

(b2) performing a first strand cDNA synthesis in the presence of an RNA-dependent DNA polymerase and a dNTP mixture to generate a pool of single-stranded cDNA molecules,

(b3) attaching a second adaptor molecule to cDNA molecules of said pool,

(c) preparing a water-in-oil emulsion by statistically distributing the cDNA molecules obtained in step (b3) into an excess of aqueous droplets of a water-in-oil emulsion so that a majority of water droplets comprise one or zero cDNA molecules; and

(d) clonally amplifying the cDNA molecules within the emulsion using polymerase chain reaction to produce an amplified population comprising different cDNA molecules present at representation frequencies that are about equal to the representation frequency of the different single-stranded RNA molecules present in the plurality.

2. The method according to claim 1, wherein the second adaptor attaching step (b3) comprises

(b3i) performing a terminal transferase reaction in the presence of one specific dNTP to create a homopolymer overhang, and

(b3ii) hybridizing a second adaptor molecule to cDNA molecules of said pool, the second adaptor molecule comprising:

a 5′

terminal part comprising a primer binding site which is either identical to or different from the 5′

terminal part of the first adaptor molecule, and

a 3′

terminal part of homopolymeric nucleotide residues, which is complementary to the homopolymer overhang.

3. The method according to claim 2, wherein

the single-stranded RNA molecules of said plurality are mRNA molecules,

the 3′

terminal part of the first adaptor molecule comprises an oligo (dT) sequence at least 5 nucleotides in length, and

the one specific dNTP is dATP.

4. The method according to claim 2, wherein the 3′

terminal part of the first adaptor molecule comprises a randomized sequence at least 5 nucleotides in length or a specific sequence for a gene or gene family.

5. The method according to claim 2, wherein the second adaptor molecule comprises a 5′

terminal part comprising a primer binding site which is identical to the 5′

terminal part of the first adaptor molecule.

6. The method according to claim 3, further comprising the step of degrading the dNTP mixture with alkaline phosphatase before the step of performing a terminal transferase reaction.

7. The method according to claim 3, further comprising the step of removing RNA molecules from the pool of single-stranded cDNA molecules with RNAse H.

8. The method according to claim 3, further comprising the step of removing the first adaptor molecule from the pool of single-stranded cDNA molecules with a 3′

-5′

exonuclease.

9. The method of either claim 7 or 8, wherein the removing step occurs when the second adaptor molecule is not present in the pool of single-stranded cDNA molecules.

10. The method of claim 8, wherein the 3′

-5′

exonuclease is DNA exonuclease I.

11. The method according to claim 1, further comprising the steps of

(e) breaking up the emulsion of clonally amplified molecules;

and

(f) sequencing the clonally amplified nucleic acid molecules.

12. The method according to claim 1, further comprising the steps of

(e) breaking up the emulsion of clonally amplified molecules;

and

(f) performing a real-time PCR reaction on the clonally amplified nucleic acid molecules.

13. The method according to claim 1, further comprising the steps of

(e) breaking up the emulsion of clonally amplified molecules;

and

(f) performing a DNA microarray analysis on the clonally amplified nucleic acid molecules.

14. The method according to claim 1, wherein the plurality of single-stranded RNA molecules is derived from less than 100 cells.

15. The method according to claim 1, wherein the plurality of single-stranded RNA molecules is derived from less than 10 cells.

16. The method according to claim 1, wherein the plurality of single-stranded RNA molecules is derived from 1 cell.

17. The method according to claim 1, wherein the 3′

terminal part of the first adaptor molecule comprises a randomized sequence at least 5 nucleotides in length or a specific sequence for a gene or gene family.

18. The method according to claim 1, wherein the second adaptor molecule comprises a 5′

terminal part comprising a primer binding site which is identical to the 5′

terminal part of the first adaptor molecule.