Amplification of nucleic acids

• US 6,582,938 B1
• Filed: 05/11/2001
• Issued: 06/24/2003
• Est. Priority Date: 05/11/2001
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Su, Xing, Ryder, Thomas B., Dong, Helin
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US09/854,317
• Time in Patent Office: 774 Days
• Field of Search: 435/6, 435/91.2, 435/91.21, 536/22.1, 536/23.1, 536/24.3
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]

Abstract

The present invention relates to the amplification of nucleic acids, preferably from mRNA. A primer and promoter are added to a target sequence to be amplified and then the target is amplified in an in vitro transcription reaction.

Claims

1. A method for the amplification of nucleic acid, said method comprising:

contacting mRNA having a poly d(A) tail with a primer consisting of SEQ ID No. 2;

generating a first cDNA strand from the mRNA strand by extending the primer by reverse transcriptase and the appropriate nucleotides under the appropriate conditions, which creates a RNA;

DNA duplex;

denaturing the RNA;

DNA duplex;

forming a double stranded DNA;

adding a promoter to the DNA by hybridizing an oligonucleotide consisting of SEQ ID No. 4 to the single stranded DNA, wherein the oligonucleotide is blocked from extension at the 3′

end;

forming a double stranded DNA promoter region by adding the appropriate reagents; and

,

producing multiple copies of RNA from the DNA strand comprising the promoter.

2. The method of claim 1, wherein any enzymes used are thermostable.

3. The method of claim 1 wherein said nucleic acid is selected from the group consisting of genomic DNA, cDNA, total RNA, poly(A)⁺ RNA, and oligonucleotides.

4. The method of claim 1 wherein said poly(A)⁺ RNA is mRNA.

5. The method of claim 1 further comprising:

contacting said multiple copies of RNA with a solid support comprising nucleic acid probes.

6. The method of claim 1 further comprising:

detecting the presence or absence of hybridization of said multiple copies of RNA to said nucleic acid probes on said solid support.

7. The method of claim 1 wherein said solid support comprising nucleic acid probes is selected from the group consisting of a nucleic acid probe array, a membrane blot, a microwell, a bead, and a sample tube.

8. The method of claim 1 wherein said nucleic acid is isolated from an eukaryotic cell or tissue.

9. The method of claim 8, wherein said eukaryotic cell or tissue is mammalian.

10. The method of claim 9 wherein said mammalian cell or tissue is human.

11. The method of claim 1 wherein said nucleic acid is isolated from a source selected from the group consisting of dissected tissue, microdissected tissue, a tissue subregion, a tissue biopsy sample, a cell sorted population, a cell culture, and a single cell.

12. The method of claim 1 wherein said nucleic acid is isolated from a cell or tissue source selected from the group consisting of brain, liver, heart, kidney, lung, spleen, retina, bone, lymph node, endocrine gland, reproductive organ, blood, nerve, vascular tissue, and olfactory epithelium.

13. The method of claim 1 wherein said nucleic acid is isolated from a cell or tissue source selected from the group consisting of embryonic and tumorigenic.

14. The method of claim 1 wherein the method involves the use of an automated machine.

15. The method of claim 14 wherein said automated machine is selected from the group consisting of a thermocycler, an integrated reaction device, and a robotic delivery system.