Kits for amplification of RNA sequence using composite primers

• US 20090036663A1
• Filed: 01/25/2008
• Published: 02/05/2009
• Est. Priority Date: 03/09/2001
• Status: Active Grant

• Current Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Original Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Inventors: Kurn, Nurith
• Granted Patent: US 7,771,946 B2
• Time in Patent Office: Days
• US Class Current: 536/24.330
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]

Abstract

The invention provides methods for isothermal amplification of RNA. The methods are particularly suitable for amplifying a plurality of RNA species in a sample. The methods employ a composite primer, a second primer and strand displacement to generate multiple copies of DNA products comprising sequences complementary to an RNA sequence of interest. In another aspect, the methods employ a single primer (which is a composite primer) and strand displacement to generate multiple copies of DNA products comprising sequences complementary to an RNA sequence of interest. In some embodiments, a transcription step is included to generate multiple copies of sense RNA of an RNA sequence of interest. The methods are useful for preparation of nucleic acid libraries and substrates for analysis of gene expression of cells in biological samples. The invention also provides compositions and kits for practicing the amplification methods, as well as methods which use the amplification products.

Claims

1. A kit for amplifying a target RNA, comprising a first composite primer and a second composite primer, wherein the first composite primer comprises an RNA portion and a 3′

DNA portion, wherein the second composite primer comprises an RNA portion and a 3′

DNA portion, and wherein the second composite primer comprises a sequence that is hybridizable to a polynucleotide comprising a complement of the first composite primer.

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

with respect to the 3′

DNA portion of the first composite primer and the RNA portion of the second composite primer is 5′

with respect to the 3′

DNA portion of the second composite primer.

3. The kit of claim 1, wherein the first composite primer is a tailed primer that comprises a 5′

portion that is not hybridizable to a target RNA under conditions wherein the first composite primer hybridizes to the target RNA.

4. The kit of claim 1, wherein the RNA portion of the first composite primer comprises at least about 5 nucleotides and the DNA portion of the first composite primer comprises at least about 5 nucleotides.

5. The kit of claim 1, wherein the 3′

DNA portion of the first composite primer comprises a random primer sequence.

6. The kit of claim 1, wherein the 3′

DNA portion of the first composite primer comprises a poly-dT sequence.

7. The kit of claim 1, wherein the RNA portion of the second composite primer comprises at least about 5 nucleotides and the DNA portion of the second composite primer comprises of at least 1 nucleotide.

8. The kit of claim 1, further comprising an enzyme that cleaves RNA from an RNA/DNA hybrid.

9. The kit of claim 1, further comprising an RNA-dependent DNA polymerase.

10. The kit of claim 1, further comprising a labeled dNTP.

11. The kit of claim 1, further comprising a set of instructions for carrying out a method comprising the steps of:

(a) producing a first primer extension product by extension of the first composite primer hybridized to a target RNA with at least one enzyme comprising RNA-dependent DNA polymerase activity;

(b) producing a second primer extension product by extension of a second primer hybridized to the first primer extension product;

(c) cleaving RNA from a complex of first and second primer extension products with at least one enzyme that cleaves RNA from an RNA/DNA hybrid; and

(d) hybridizing a second composite primer to the second primer extension product and extending the second composite primer with at least one enzyme comprising DNA-dependent DNA polymerase activity,

whereby said first primer extension product is displaced, RNA is cleaved from the second composite primer and another second composite primer hybridizes such that primer extension and strand displacement are repeated; and

whereby multiple copies of a polynucleotide sequence complementary to the RNA sequence of interest are generated.

12. The kit of claim 2, wherein the 5′

RNA portion of the first composite primer is adjacent to the 3′

DNA portion of the first composite primer and the 5′

RNA portion of the second composite primer is adjacent to the 3′

DNA portion of the second composite primer.

13. The kit of claim 2, wherein the first composite primer consists of the 5′

RNA portion and a 3′

DNA portion.

14. The kit of claim 2, wherein the first composite primer is a tailed primer that comprises a 5′

portion that is not hybridizable to the target RNA under conditions in which the first primer hybridizes to the target RNA.

15. The kit of claim 3, wherein the RNA portion of the first composite primer comprises at least about 5 nucleotides and the DNA portion of the first composite primer comprises at least about 5 nucleotides.

16. The kit of claim 3, wherein the RNA portion of the second composite primer comprises at least about 5 nucleotides and the DNA portion of the second composite primer comprises of at least 1 nucleotide.

17. The kit of claim 3, wherein the 3′

DNA portion of the first composite primer comprises a poly-dT sequence.

18. The kit of claim 3, further comprising a set of instructions for carrying out a method comprising the steps of:

(a) producing a first primer extension produced by extension of the first composite primer hybridized to a target RNA with at least one enzyme comprising RNA-dependent DNA polymerase activity;

(b) producing a second primer extension product by extension of a second primer hybridized to the first primer extension product;

(c) cleaving RNA from a complex of first and second primer extension products with at least one enzyme that cleaves RNA from an RNA/DNA hybrid; and

(d) hybridizing a second composite primer to the second primer extension product and extending the second composite primer with at least one enzyme comprising DNA-dependent DNA polymerase activity,

whereby said first primer extension product is displaced, RNA is cleaved from the second composite primer and another second composite primer hybridizes such that primer extension and strand displacement are repeated; and

whereby multiple copies of a polynucleotide sequence complementary to the RNA sequence of interest are generated.

19. The kit of claim 3, wherein the first composite primer comprises a random primer sequence.

20. The kit of claim 4, wherein the RNA portion of the first composite primer consists of about 5 to about 50 nucleotides and the DNA portion of the first composite primer consists of about 5 to about 20 nucleotides.

21. The kit of claim 6, wherein the DNA portion of the first composite primer comprises at least 1 random nucleotide at the 3′

end.

22. The kit of claim 7, wherein the RNA portion of the second composite primer consists of about 5 to about 50 nucleotides and the DNA portion of the second composite primer consists of about 1 to about 20 nucleotides.

23. The kit of claim 8, wherein the enzyme that cleaves RNA from an RNA/DNA hybrid is RNaseH.

24. The kit of claim 12, wherein the RNA portion of the second composite primer comprises at least about 5 nucleotides and the DNA portion of the second composite primer comprises of at least 1 nucleotide.

25. The kit of claim 12, wherein the composite primer is a tailed primer that comprises a 5′

portion that is not hybridizable to a target RNA under conditions wherein the composite primer hybridizes to the target RNA.

26. The kit of claim 12, wherein the RNA portion of the first composite primer comprises at least about 5 nucleotides and the DNA portion of the first composite primer comprises at least about 5 nucleotides.

27. The kit of claim 12, wherein the first composite primer consists of the 5′

RNA portion and a 3′

DNA portion.

28. The kit of claim 17, wherein the DNA portion of the first composite primer comprises at least 1 random nucleotide at the 3′

end.