One-step reverse transcription template-switching pcr

A method of producing a nucleic acid sample that is amplified based on at least a part of a region of a target RNA, the method comprising the steps of:

• a) mixing the target RNA, a reagent required for reverse transcription, a reagent required for template switching, and a reagent required for a polymerase chain reaction and subjecting the mixture to a condition under which reverse transcription occurs to provide a cDNA comprising a nucleic acid sequence corresponding to the target RNA and a template switching oligonucleotide;
• and
• b) subjecting the cDNA obtained in step a) to a condition under which a polymerase chain reaction occurs;
• wherein the reagent required for a polymerase chain reaction comprises a modified oligonucleotide primer designed to have a primer function that is partially or completely blocked in step a) and designed to have blocking of the primer function cleared in step b).

A kit for amplifying at least a part of a region of a target RNA, the kit comprising:

• i) a reagent required for reverse transcription;
• ii) a reagent required for template switching;
• iii) a reagent required for a polymerase chain reaction using a modified oligonucleotide primer;
• and
• iv) optionally a user manual;
• characterized in that the reagents of i) to iii) and the modified oligonucleotide primer are all mixed in a reaction system as of the initiation of a reaction, wherein the modified oligonucleotide primer is designed to have a primer function that is partially or completely blocked under a condition where reverse transcription occurs and designed to have blocking of the primer function cleared under a condition where a polymerase chain reaction occurs.