CLONAL PRE-AMPLIFICATION IN EMULSION

• US 20120010086A1
• Filed: 06/27/2011
• Published: 01/12/2012
• Est. Priority Date: 07/07/2010
• Status: Active Grant

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Roche Molecular Systems (Roche Holding AG)
• Inventors: Froehlich, Thomas, Heckel, Tobias
• Granted Patent: US 9,650,629 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• 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 comprising the steps of

(a) 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 a majority of water droplets comprises one or none of the plurality of different nucleic acid molecules, and

(d) clonally amplifying the plurality of different nucleic acid molecules.

2. The method according to claim 1, wherein the different nucleic acid molecules are single stranded molecules.

3. The method according to claim 1, wherein the different nucleic acid molecules are RNA molecules.

4. The method according to claim 1, wherein the different nucleic acid molecules are polyadenylated RNA molecules.

5. The method according to claim 1, wherein the different nucleic acid molecules are mRNA molecules.

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

(b1) hybridizing a first single stranded adaptor nucleic acid molecule to the plurality of different nucleic acid molecules, the adapter molecule comprising

a 5′

terminal part representing a primer binding site, and

a 3′

terminal part which is either an oligo dT sequence of at least 5 nucleotides in length, an essentially randomized sequence of at least 5 nucleotides in length, or a gene family specific sequence,

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

(b3) attaching a second single stranded adaptor molecule to the pool of single stranded cDNAs.

7. The method according to claim 6, wherein step (b3) comprises

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

(b3ii) hybridizing a second single stranded adaptor molecule to the pool of single stranded cDNAs, the second single stranded adapter molecule further comprising

a 5′

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

terminal part of the first single stranded adaptor molecule and

a 3′

terminal part of homopolymeric nucleotide residues, which is complementary to the homopolymer overhang created in step (b3i).

8. The method according to claim 7, wherein

the plurality of different nucleic acid molecules are mRNA molecules,

the 3′

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

the specific dNTP in step (b4) is dATP.

9. The method according to claim 8, further comprising the step of digesting the mRNA molecules by means of incubation with RNAse H either between steps (b2) and (b3i) or during step (b3i).

10. The method according to claim 8, further comprising the step of degrading the dNTP mixture by means of incubation with alkaline phosphatase between steps (b2) and (b3i).

11. The method according to claim 8, further comprising the step of degrading the first single stranded adaptor nucleic acid by means of incubation with a 3′

-5′

exonuclease, which is preferably DNA exonuclease I between steps (b2) and step (b3i).

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

(e) breaking up the emulsion and

(f) sequencing the clonally amplified plurality of different nucleic acid molecules.

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

(e) breaking up the emulsion and

(f) performing a real time PCR reaction.

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

(e) breaking up the emulsion and

(f) performing a DNA microarray analysis.

15. The method according to claim 1, wherein the plurality of different nucleic acid molecules is derived from less than 100 cells.

16. The method according to claim 1, wherein the plurality of different nucleic acid molecules is derived from less than 10 cells.

17. The method according to claim 1, wherein the plurality of different nucleic acid molecules is derived from 1 cell.

18. A kit comprising

a first single stranded adaptor nucleic acid molecule comprising

a 5′

terminal part representing a primer binding site and

a 3′

terminal part which is either an oligo dT sequence of at least 5 nucleotides in length, an essentially randomized sequence of at least 5 nucleotides in length or a gene specific sequence, and

a second single stranded adaptor molecule comprising

a 5′

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

terminal part of the first single stranded adaptor molecule, and

a 3′

terminal part of homopolymeric nucleotide residues.

19. The kit according to claim 18 further comprising at least a terminal transferase and optionally a reverse transcriptase and/or thermostable DNA polymerase.

20. The kit according to claim 18, wherein the 3′

terminal part of the first single stranded adaptor molecule comprises an oligo dT sequence of at least 5 nucleotides in length.