Amplification of RNA sequences using composite RNA-DNA primers and strand displacement

• US 7,351,557 B2
• Filed: 06/08/2005
• Issued: 04/01/2008
• Est. Priority Date: 03/09/2001
• Status: Expired due to Fees

• Current Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Original Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Inventors: Kurn, Nurith
• Primary Examiner(s): Horlick; Kenneth R.
• Application Number:
  US11/148,593
• Publication Number:
  US 20060014182A1
• Time in Patent Office: 1,028 Days
• Field of Search: 435/91.1, 435/91.2
• US Class Current: 435/91.1
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• 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]
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence

Abstract

The invention provides methods for linear amplification of RNA that has an RNA sequence of interest. The methods are based on using a first DNA primer with a 3′ portion that is complementary to the RNA and a 5′ portion that is not complementary to the RNA in that region. The primer and an RNA-dependent DNA polymerase are used to make a DNA-RNA complex from the RNA. The RNA is cleaved from the complex with an enzyme, then a second primer and a DNA-dependent DNA polymerase are used to make double stranded DNA. The double stranded DNA is then denatured, and an RNA-DNA composite primer, a DNA-dependent DNA polymerase, and strand displacement are used to isothermally produce multiple copies of the complementary sequence to the RNA sequence of interest.

Claims

1. A method of generating multiple copies of the complementary sequence of an RNA sequence of interest, said method comprising:

(a) extending a first DNA primer hybridized to a target RNA with an RNA-dependent DNA polymerase, wherein said first primer comprises a 3′

portion which hybridizes to the target RNA and a 5′

portion which does not hybridize to the target RNA, whereby a complex comprising a first primer extension product and the target RNA is produced;

(b) cleaving RNA in the complex of step (a) with an enzyme that cleaves RNA from an RNA/DNA hybrid;

(c) extending a second primer hybridized to the first primer extension product with a DNA-dependent DNA polymerase whereby a complex comprising the first primer extension product and a second primer extension product is produced;

(d) denaturing the complex of step (c);

(e) hybridizing, a composite primer to a single stranded second primer extension product resulting from step (d), wherein the composite primer comprises an RNA portion and a 3′

DNA portion, wherein the composite primer hybridizes to a sequence in said single stranded primer extension product which is complementary to a 5′

portion of the first DNA primer;

(f) extending the composite primer with a DNA-dependent DNA polymerase, whereby a complex comprising a primer extension product and the second primer extension product is formed;

(g) cleaving RNA in the complex of step (f) with an enzyme that cleaves RNA from an RNA/DNA hybrid, such that another composite primer hybridizes to the second primer extension product and repeats primer extension by strand displacement,

whereby multiple copies of the complement of the RNA sequence of interest are produced.

2. The method of claim 1, wherein the RNA portion of the composite primer is 5′

with respect to the 3′

DNA portion.

3. The method of claim 2, wherein the 5′

RNA portion is adjacent to the 3′

DNA portion.

4. The method of claim 1, wherein the first primer comprises a portion of the composite primer sequence.

5. The method of claim 1, wherein the composite primer comprises a portion hybridizable to the complement of the 5′

portion of the first primer.

6. The method of claim 1, wherein the target RNA is mRNA.

7. The method of claim 1, wherein said method comprises generating multiple copies of two or more different sequences of interest.

8. The method of claim 1, wherein the enzyme that cleaves RNA from an RNA/DNA hybrid is RNase H.

9. The method of claim 1, wherein the RNA-dependent DNA polymerase and a DNA-dependent DNA polymerase are the same enzyme.

10. The method of claim 1, wherein the RNA-dependent DNA polymerase and enzyme that cleaves RNA from an RNA/DNA hybrid are the same enzyme.

11. The method of claim 1, wherein a DNA-dependent DNA polymerase and enzyme that cleaves RNA from an RNA/DNA hybrid are the same enzyme.

12. The method of claim 1, wherein the DNA-dependent DNA polymerase in step (c), the DNA-dependent DNA polymerase in steps (f) and (g), the RNA-dependent DNA polymerase and the enzyme that cleaves RNA from an RNA/DNA hybrid are the same enzyme.

13. The method of claim 1, wherein the DNA polymerase in steps (f) and (g) incorporates dNTP'"'"'s into the copies of the complement of the RNA sequence, and at least one type of dNTP used is a labeled dNTP, whereby labeled copies are generated.

14. The method of claim 1, wherein the DNA-dependent DNA polymerase in step (c) is the same enzyme as the DNA-dependent DNA polymerase in steps (f) and (g).