Splint nucleic acid molecule for cyclizing single-stranded nucleic acid molecule and use thereof

A method for cyclizing a single-stranded nucleic acid molecule, comprising:

• allowing a reaction mixture containing a splint nucleic acid molecule, a single-stranded nucleic acid molecule and a ligase to be under a condition suitable for ligation, so as to obtain a cyclized single-stranded nucleic acid molecule,
• wherein the splint nucleic acid molecule is for cyclizing the single-stranded nucleic acid molecule and consists of a 5′ terminal fragment and a 3′ terminal fragment,
• wherein the single-stranded nucleic acid molecule comprises:
• an insert fragment;
• a first adaptor, connected to a 5′ terminal of the insert fragment;
• and
• a second adaptor, connected to a 3′ terminal of the insert fragment,
• wherein the 5′ terminal fragment of the splint nucleic acid molecule is adapted to form a first complementary region between the 5′ terminal fragment of the splint nucleic acid molecule and at least a part of the first adaptor, and
• the 3′ terminal fragment of the splint nucleic acid molecule is adapted to form a second complementary region between the 3′ terminal fragment of the splint nucleic acid molecule and at least a part of the second adaptor,
• wherein the length of the first complementary region is equal to the length of the 5′ terminal fragment of the splint nucleic acid molecule, and the length of the second complementary region is equal to the length of the 3′ terminal fragment of the splint nucleic acid molecule,
• wherein the length of the first complementary region is different from the length of the second complementary region, and
• the method specifically comprises steps of:
• (i) allowing the reaction mixture to be at a first temperature for a first predetermined time, wherein the first temperature is suitable to form the first complementary region;
• (ii) allowing the reaction mixture obtained in step (i) to be at a second temperature for a second predetermined time, wherein the second temperature is suitable to form the second complementary region;
• (iii) allowing the reaction mixture obtained in step (ii) to be at a third temperature for a third predetermined time, wherein the third temperature is suitable to unwind at least one of the first complementary region and the second complementary region;
• (iv) subjecting the reaction mixture obtained in step (iii) to steps (i) and (ii) in sequence, and
• (v) subjecting the reaction mixture obtained in step (iv) to steps (iii), (i) and (ii) in sequence for at least one cycle,
• wherein the first temperature is higher than the second temperature.