Compositions and methods for cDNA synthesis

• US 7,470,515 B2
• Filed: 09/29/2005
• Issued: 12/30/2008
• Est. Priority Date: 09/03/2002
• Status: Expired due to Term

• Current Assignee: Qiagen Beverly, Inc. d/b/a Enzymatics Inc. (Qiagen NV)
• Original Assignee: Quanta Biosciences Incorporated
• Inventors: Rashtchian, Ayoub, Schuster, David M.
• Primary Examiner(s): Chunduru; Suryaprabha
• Application Number:
  US11/237,922
• Publication Number:
  US 20060068436A1
• Time in Patent Office: 1,188 Days
• Field of Search: 435/6, 435/91.21, 536/24.33, 536/22.1
• US Class Current: 435/6.18
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2527/143 Concentration of primer or probe

Abstract

Methods for making cDNA molecules, for amplification of RNA by PCR and for preparation of cDNA libraries are provided. Kits for making cDNA molecules also are provided. Compositions are also provided comprising mixtures of reagents, including reverse transcriptases, buffers, cofactors and other components, suitable for immediate use in conversion of RNA into cDNA and RT PCR without dilution or addition of further components. These compositions are useful, alone or in the form of kits, for cDNA synthesis or nucleic acid amplification (e.g., by the Polymerase Chain Reaction) or for any procedure utilizing reverse transcriptases in a variety of research, medical, diagnostic, forensic and agricultural applications.

Claims

1. A method for first strand cDNA synthesis comprising contacting one or more RNA molecules with a reagent mixture, wherein said reagent mixture comprises:

(a) at least one reverse transcriptase;

(b) a combination of primers wherein said combination comprises;

(i) an effective amount of a mixture of random primers, wherein said random primers are present in a concentration of between about 5 ng/μ

l and about 20 ng/μ

l;

(ii) an effective amount of oligo(dT), wherein said oligo(dT) is present in a concentration less than about 2 μ

M,

and incubating the resulting solution under conditions suitable for first strand cDNA synthesis wherein said random primers and said oligo (dT) prime cDNA synthesis in a single step.

2. The method according to claim 1, wherein said oligo(dT) is present in a concentration of between about 25 nM and about 2 μ

M.

3. The method according to claim 1, wherein said random primers are between 5 and 10 nucleotides long.

4. The method according to claim 1, wherein said random primers are random hexamers.

5. The method according to claim 1, wherein said oligo(dT) consists essentially of between about 12 and about 25 dT residues.

6. The method according to claim 1, wherein said oligo(dT) is oligo(dT)_12-18(SEQ ID NO:

26) or oligo(dT)₂₀(SEQ ID NO;

     25).

7. The method according to claim 1, wherein said at least one reverse transcriptase is a viral reverse transcriptase.

8. The method according to claim 1, wherein said reverse transcriptase is selected from the group consisting of AMV RT, RSV RT, MMLV RT, HlV RT, EIAV RT, RAV2 RT, TTH DNA polymerase, C.hydrogenoformans DNA polymerase, SUPERSCRIPT®

I RT, SUPERSCRIPT®

I RT, THERMOSCRIPT™

RT MMLV and Rnase H⁻

mutants thereof

9. The method of claim 1,further comprising:

an effective amount of Li ion.

10. The method according to claim 9, wherein said reverse transcriptase is a viral reverse transcriptase.

11. The method according to claim 10, wherein said viral reverse transcriptase is selected from the group consisting of AMV RT, RSV RT, MMLV RT, HIV RT, EIAV RT, RAV2 RT, SUPERSCRIPT®

II RT, SUPERSCRIPT®

I RT, THERMOSCRIPT™

RT MMLV and Rnase H⁻

mutants thereof.

12. The method according to claim 9, wherein said Li ion is present in a concentration of between about 5 mM to about 200 mM.

13. The method according to claim 12, wherein said buffer further comprises at least one additional monovalent cation in a concentration between about 20 mM and 200 mM, wherein said monovalent cation is selected from the group consisting of Na, K, and NH4, and wherein the total concentration of said Li ion and said further monovalent cation is less than or equal to about 200 mM.

14. The method according to claim 13, wherein said additional monovalent cation is K.

15. The method according to claim 1, wherein said oligo(dT) is an anchored oligo(dT).

16. The method according to claim 15, wherein the 3′

nucleotide of said anchored oligo(dT) is dA.

17. The method according to claim 15, wherein the 3′

nucleotide of said anchored oligo(dT) is dC.

18. The method according to claim 15, wherein the 3′

nucleotide of said anchored oligo(dT) is dG.

19. The method according to claim 15, wherein the 3′

end of said anchored oligo(dT) is selected from the group consisting of dAdA, dAdG, dAdC, dGdG, dGdA, dGdC, dCdC, dCdA, and dCdG.

20. The method according to claim 1, wherein said oligo(dT) comprises at least one dU residue.