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

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.

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.