Preparation and use of single-stranded transcription substrates for synthesis of transcription products corresponding to target sequences

1. A method for making a transcription product having a sequence that is identical to at least a portion of a target sequence in a target nucleic acid consisting of RNA, the method comprising the steps of:

(a) providing a sense promoter primer, the sense promoter primer exhibiting, at its 3′-end, a sequence complementary to the 3′-end of the target sequence, the sense promoter primer further exhibiting, at its 5′-end, a sense transcription promoter sequence;

(b) annealing the sense promoter primer to the target nucleic acid and primer-extending the sense promoter primer with an RNA-dependent DNA polymerase using target nucleic acid as a template to generate linear sense-promoter-containing first-strand cDNA;

(c) removing the target nucleic acid;

(d) ligating the first-strand cDNA to itself with a ligase, whereby the 5′-end is covalently joined to the 3′-end to obtain circular first-strand cDNA containing the sense transcription promoter sequence;

(e) obtaining a circular transcription substrate; and

(f) admixing the transcription substrate with an RNA polymerase and NTPs and incubating under conditions wherein a transcription product having a sequence that is identical to at least a portion of a target sequence is synthesized.

2. The method of claim 1 , additionally comprising the step of cleaving the circular transcription substrate at a site that is 3′-of the promoter sequence and 5′-of the target-complementary sequence to generate a linear transcription substrate.

3. The method of claim 1 , wherein the target nucleic acid in the sample is mRNA.

4. The method of claim 1 , wherein the sense promoter primer is selected from the group consisting of:

(i) an oligo(dT) promoter primer;

(ii) an anchored oligo(dT) promoter primer;

(iii) a specific-sequence promoter primer that is complementary to a specific sequence in the target nucleic acid sequence; and

(iv) a random sequence promoter primer that exhibits a random sequence at its 3′-end.

5. The method of claim 1 , wherein the sense promoter primer additionally comprises one or more transcription termination sequences between the target-complementary sequence at its 3′-end and the sense promoter sequence in its 5′-end portion.

6. The method of claim 1 , wherein the sense promoter primer additionally comprises a transcription initiation sequence 5′-of the sense promoter sequence.

7. The method of claim 1 , wherein, between the target-complementary sequence at its 3′-end and the sense promoter sequence in its 5′-end portion, the sense promoter primer additionally comprises one or more sequences or genetic elements selected from among one or more origins of replication, one or more sequences that encode a selectable or screenable marker, one or more sequences that can be recognized and used by a transposase for in vitro or in vivo transposition, and one or more sites that are recognized by a recombinase.

8. The method of claim 2 , wherein the sense promoter primer has a dUMP nucleotide between the target-complementary sequence at its 3′-end and the sense promoter sequence in its 5′-end portion.

9. The method of claim 8 , wherein the circular transcription substrate is linearized by treatment with uracil-N-glycosylase (UNG) and endonuclease IV (endo IV).

10. The method of claim 1 , wherein the ligase used in step (d) for said joining is a thermostable RNA ligase derived from phage TS2126.

11. The method of claim 1 , wherein the method is performed in a stepwise fashion by purifying the reaction products by removing reaction components and/or inactivating enzymes from one set of reactions prior to proceeding to the next set of reactions.

12. The method of claim 11 , wherein the linear sense promoter-containing first-strand cDNA reaction product is purified prior to the step of circularizing with a ligase.

13. A method for generating linear first-strand cDNA complementary to a target sequence in a target nucleic acid wherein its 3′ and 5′ ends exhibit sequences that are not complementary to the target sequence, the method comprising:

(1) primer extending a primer with a DNA polymerase using target nucleic acid as a template to generate linear first-strand cDNA that is complementary to the target sequence, wherein the primer comprises

(i) a 5′-end portion that is not complementary to the target nucleic acid,

(ii) a 3′-end portion that is complementary to the target nucleic acid, and

(iii) a cleavage site within its 5′-end portion;

(2) purifying the linear first-strand cDNA reaction products by removing reaction components and/or inactivating enzymes from the DNA polymerase primer extension reaction of step (1);

(3) circularizing the linear first-strand cDNA generated in step (1) with a ligase under ligation conditions to generate circular first-strand cDNA;

(4) purifying the circular first-strand cDNA by removing reaction components and/or inactivating enzymes from the ligase reaction of step (3);

(5) linearizing the circular first-strand cDNA generated in step (3) at the cleavage site to generate linear first-strand cDNA that exhibits the sequence 3′-of the cleavage site of the primer in its 5′-end portion and the sequence 5′-of the cleavage site of the primer in its 3′-end portion; and

(6) using the linear first-strand cDNA generated in step (5) for sequencing of the target nucleic acid.

14. The method of claim 13 , wherein the ligase for circularizing is the RNA ligase derived from phage TS2126.

15. The method of claim 13 , wherein the cleavage site in the primer comprises a dUMP nucleotide.

16. The method of claim 15 , wherein said step of linearizing the circular first-strand cDNA comprises treating with uracil-N-glycosylase (UNG) and endonuclease IV (endo IV).

17. The method of claim 13 , wherein the target nucleic acid is RNA and the primer is selected from the group consisting of:

(i) an oligo(dT) primer;

(ii) an anchored oligo(dT) primer;

(iii) a specific-sequence primer that is complementary to a specific sequence in the target nucleic acid sequence; and

(iv) a random sequence primer that exhibits a random sequence at its 3′-end.

18. A method comprising:

(a) providing a sample that contains RNA target nucleic acid;

(b) providing a primer, the primer comprising a 5′-end portion that exhibits a sequence that is not complementary to the target nucleic acid and a 3′-end portion that is complementary to the target nucleic acid, wherein the primer contains a cleavage site between the 5′-end portion and the 3′-end portion;

(c) annealing the primer to the target nucleic acid so as to form a target nucleic acid-primer complex;

(d) contacting the target nucleic acid-primer complex with a DNA polymerase under polymerization reaction conditions so as to synthesize first-strand cDNA that is complementary to a sequence in the target nucleic acid;

(e) obtaining the first-strand cDNA by removing the target nucleic acid;

(f) ligating the first-strand cDNA under ligation conditions in the absence of a ligation splint using the RNA ligase derived from phage TS2126, which catalyzes non-homologous ligation of ssDNA, to obtain circular first-strand cDNA that exhibits the sequences of the 5′-end portion and of the 3′-end portion of the primer; and

(g) linearizing the circular first-strand cDNA at the cleavage site to obtain linear first-strand cDNA that exhibits the sequence of the 3′-end portion of the primer at its 5′ end and the 5′-end portion of the primer at its 3′ end.

19. The method of claim 18 , wherein the cleavage site is a dUMP residue that can be cleaved using uracil-N-glycosylase and endonuclease IV.

20. The method of claim 18 , further comprising sequencing the linear first-strand cDNA that exhibits the sequence of the 3′-end portion of the primer at its 5′ end and the 5′-end portion of the primer at its 3′ end.

21. The method of claim 18 , further comprising detecting the linear first-strand cDNA that exhibits the sequence of the 3′-end portion of the primer at its 5′ end and the 5′-end portion of the primer at its 3′ end, which detecting is indicative of the presence of the target nucleic acid.

22. The method of claim 18 , further comprising quantifying the amount of target nucleic acid in the sample by comparing the amount of the linear first-strand cDNA that exhibits the sequence of the 3′-end portion of the primer at its 5-end and the 5′-end portion of the primer at its 3′-end which is amplified from the sample to the amount of product which is amplified from of a reference sample that has a known quantity of the target nucleic acid.

23. The method of claim 1 , wherein said sense promoter primer provided in step (a) exhibits a sense transcription promoter sequence of a double-stranded transcription promoter and said obtaining a circular transcription substrate in step (e) further comprises annealing an oligonucleotide to the circular first-strand cDNA containing the sense transcription promoter sequence under conditions wherein a circular transcription substrate having a double-stranded promoter is obtained.

24. The method of claim 1 , wherein said sense promoter primer provided in step (a) exhibits a sense transcription promoter sequence of a single-stranded transcription promoter and the circular first-strand cDNA containing the sense transcription promoter sequence obtained in step (d) is a circular transcription substrate.