Nucleic acid amplification using an RNA polymerase and DNA/RNA mixed polymer intermediate products

1. A method of amplifying a target nucleic acid in an aqueous solution with a first and a second primer, said method comprising:

i.) transcribing an intermediate duplex with a phage-encoded RNA polymerase to form a sense transcription product having a 5′ end and a 3′ end,

wherein said intermediate duplex comprises a double-stranded nucleic acid, wherein said double-stranded DNA molecule comprises a first and a second strand,

wherein said first strand comprises in the following order from 5′ to 3′:

a phage-encoded RNA polymerase recognition sequence,

a first spacer sequence comprising a sequence of from 12 to 20 nucleotides that consists of one nucleotide type or two different nucleotide types, and

a first target complementary sequence which can bind to a segment of said target nucleic acid,

wherein said second strand comprises in the following order from 5′ to 3′:

a second target complementary sequence which can bind to a segment of said target nucleic acid,

a second spacer sequence comprising a sequence of from 12 to 20 nucleotides that consists of one nucleotide type or two different nucleotide types, and

a phage-encoded RNA polymerase recognition sequence,

wherein said transcribing takes place in the presence of Mn++, with all four dNTPs, and with those rNTPs represented in said first spacer sequence;

ii.) hybridizing said second primer to said sense transcription product to form a second primer-sense transcription product complex,

wherein said second primer comprises in the following order from 5′ to 3′:

a phage-encoded RNA polymerase recognition sequence,

said second spacer sequence, and

said second target complementary sequence which can bind to a 3′ segment of said target nucleic acid;

iii.) extending said second primer-sense transcription product complex with a Reverse Transcriptase that lacks RNAseH activity to form a first amplification duplex;

iv.) transcribing said first amplification duplex with a phage-encoded RNA polymerase, in the presence of Mn++, with all four dNTPs, and with those rNTPs represented in said second spacer sequence, to form an antisense transcription product;

v.) hybridizing said first primer to said antisense transcription product to form a first primer-antisense transcription product complex,

wherein said first primer comprises in the following order from 5′ to 3′:

a phage-encoded RNA polymerase recognition sequence,

said first spacer sequence, and

said first target complementary sequence which can bind to a 5′ segment of said target nucleic acid;

vi.) extending said first primer-antisense transcription product complex with a Reverse Transcriptase that lacks RNAseH activity to form a second amplification duplex; and

vii.) transcribing said second amplification duplex with a phage-encoded RNA polymerase, in the presence of Mn++, with all four dNTPs, and with those rNTPs represented in said first spacer sequence to form said sense transcription product.

2. The method of claim 1 , wherein the method further comprises repetitively carrying out steps i to vii.

3. The method of claim 1 , wherein said first or said second spacer sequence comprises a nucleotide sequence having the formula (XY)n,

wherein n is from 6 to 10,

wherein X and Y are independently selected from the group consisting of an adenine nucleotide, a guanine nucleotide, a cytosine nucleotide, and a thymidine nucleotide,

wherein X and Y are not the same.

4. The method of claim 3 , wherein X is an adenine nucleotide and Y is a guanine nucleotide.

5. The method of claim 4 , wherein n is 9.

6. The method of claim 4 , wherein the rNTPs are rATP and rGTP.

7. The method of claim 1 , wherein said first or said second spacer sequence comprises a nucleotide sequence having the formula (X)n,

wherein n is from 12 to 20,

wherein X is selected from the group consisting of an adenine nucleotide, a guanine nucleotide, a cytosine nucleotide, and a thymidine nucleotide.

8. The method of claim 7 , wherein n is 18.

9. The method of claim 1 , wherein said sense and antisense transcription products comprise a nucleic acid strand comprising both ribonucleotides and deoxynbonucleotides.

10. The method of claim 1 , wherein said first and said second amplification duplexes consist of deoxyribonucleotides and ribonucleotides.

11. The method of claim 1 , wherein said method is carried out at a single temperature.

12. The method of claim 1 , wherein said method is carried out at a single temperature of between 25° C. and 55° C.

13. The method of claim 1 , wherein the method is carried out at a single temperature of greater than 50° C.

14. The method of claim 1 , wherein said intermediate duplex comprises a double-stranded DNA comprising one complete primer sequence followed by the entire sequence that is to be amplified.

15. The method of claim 1 , wherein said intermediate duplex is formed from double-stranded DNA, single-stranded DNA, or RNA.

16. The method of claim 1 , wherein said intermediate duplex is formed by the process comprising the following steps of:

denaturing a double-stranded DNA target to form an upper strand and a lower strand;

hybridizing said first primer to said lower strand to form a first primer-lower strand complex;

extending said first primer-lower strand complex with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to form a first long sense strand product-lower strand complex;

denaturing said first long sense strand product-lower strand complex into a first long sense strand product and said lower strand;

hybridizing said second primer to said first long sense strand product to form a second primer-first long sense strand product; and

extending said first primer-first long antisense strand product with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to yield said intermediate duplex.

17. The method of claim 1 , wherein said intermediate duplex is formed by the process comprising the following steps of:

denaturing a double-stranded DNA target to form an upper strand and a lower strand;

hybridizing said first primer to said lower strand to form a first primer-lower strand complex;

extending said first primer-lower strand complex with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to form a first long sense strand product-lower strand complex, wherein said first long sense strand product has a 5′ and a 3′ end;

displacing said first sense strand product from said lower strand by:

hybridizing a bumper oligonucleotide to a subsequence on said lower strand adjacent to said 5′ end of said first sense strand product on the first sense strand product-lower strand complex;

extending said bumper oligonucleotide with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase, thereby displacing said first sense strand product;

hybridizing said second primer to said first long sense strand product to form a second primer-first long sense strand product; and

extending said first primer-first long antisense strand product with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to yield said intermediate duplex.

18. The method of claim 1 , wherein said intermediate duplex is formed by the process comprising the following steps of:

hybridizing said second primer to a target RNA molecule to form a second primer-RNA template complex;

extending said second primer-target RNA molecule complex with a Reverse Transcriptase that lacks RNAseH activity or a DNA Polymerase to form a first long antisense strand product-template complex, wherein said first long antisense strand product has a 5′ and a 3′ end;

displacing said first long antisense strand product from said target RNA molecule by:

hybridizing a bumper oligonucleotide to a subsequence on said target RINA molecule adjacent to said 5′ end of said first sense strand product on the first sense strand product-lower strand complex;

extending said bumper oligonucleotide with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase, thereby displacing said first long antisense strand product;

hybridizing said first primer to said first long antisense strand product to form a first primer-first long antisense strand product complex; and

extending said first primer-first long antisense strand product with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to yield said intermediate duplex.

19. The method of claim 1 , wherein said intermediate duplex is formed by the process comprising the following steps of:

hybridizing said second primer to a single-stranded target RNA molecule to form a second primer-RNA template complex;

extending said second primer-RNA template complex with a Reverse Transcriptase that lacks RNAseH activity or a DNA Polymerase to form a first long antisense strand product-template complex;

denaturing said first long antisense strand product-RNA template complex into a first long antisense strand product and said single-stranded RNA molecule;

hybridizing said first primer to said first long antisense strand product to form a first primer-first long antisense strand product complex; and

extending said first primer-first long antisense strand product with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to yield said intermediate duplex.

20. The method of claim 1 , wherein said phage-encoded RNA polymerase is polymerase selected from the group consisting of: a T7 RNA polymerase, a T4 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase and a K11 RNA polymerase.

21. The method of claim 20 , wherein said phage-encoded RNA polymerase is a mutant phage-encoded RNA polymerase that is competent to incorporate dNTPs into a template nucleic acid.

22. The method of claim 21 , wherein said phage-encoded RNA polymerase is a T7 RNA polymerase.

23. The method of claim 22 , wherein said T7 RNA polymerase contains a Y639F mutation.

24. The method of claim 22 , wherein said T7 RNA polymerase contains a S641A mutation.

25. The method of claim 22 , wherein said T7 RNA polymerase contains at least two mutations.

26. The method of claim 1 , wherein said Mn++ is present in a concentration of between 10 μM to 20 mM.

27. The method of claim 26 , wherein said concentration is 10 mM.

28. The method of claim 1 , wherein said target nucleic acid is single-stranded DNA.

29. The method of claim 1 , wherein the target nucleic acid is comprised of RNA.

30. The method of claim 1 , further detecting said sense transcription product, said antisense transcription product, said first amplification duplex, or said second amplification duplex,

wherein said detecting comprises hybridizing a detection oligonucleotide comprising a detectable moiety, wherein said detection oligonucleotide is complementary to a subsequence of said sense transcription product, said antisense transcription product, said first amplification duplex, or said second amplification duplex.