Patent ID: 6974670

Claim:
A method of making a single-stranded nucleic acid molecule having stem and loop formations at the 5′ and 3′ ends thereof, the method comprising: annealing a first oligonucleotide primer to a sample single-stranded nucleic acid molecule, the first oligonucleotide primer comprising a 3′ end portion which anneals to the sample single-stranded nucleic acid molecule and a 5′ end portion comprising substantially the same nucleotide sequence as an arbitrary region of the sample single-stranded nucleic acid molecule; extending the first oligonucleotide primer from its 3′ end, using a suitable polymerase, to form a first single-stranded nucleic acid molecule comprising a 5′ end portion comprising a first region and a first complementary region located 5′ terminal which, under suitable conditions, anneal to one another to form a loop; displacing the first single-stranded nucleic acid molecule from the sample single-stranded nucleic acid molecule; annealing a second oligonucleotide primer to the first single-stranded nucleic acid molecule, the second oligonucleotide primer comprising a 3′ end portion which anneals to the first single-stranded nucleic acid molecule and a 5′ end portion comprising substantially the same nucleotide sequence as an arbitrary region of the first single-stranded nucleic acid molecule; extending the second oligonucleotide primer from its 3′ end, using a suitable polymerase, to form a second single-stranded nucleic acid molecule comprising (i) a 3′ end portion complementary to the 5′ end portion of the first single-stranded nucleic acid molecule, the 3′ end portion comprising the first region located 3′ terminal and the first complementary region which, under suitable circumstances, anneal to one another to form a first loop, and (ii) a 5′ end portion comprising a second complementary region located 5′ terminal and a second region which, under suitable circumstances, anneal to one another to from a second loop; and displacing the second single-stranded nucleic acid molecule from the first single-stranded nucleic acid molecule, whereby the second single-stranded nucleic acid molecule assumes a conformation with a stem and loop formation formed at both the 3′ end portion and the 5′ end portion; wherein either (i) said displacing the first single-stranded nucleic acid molecule is carried out beginning from where the 3′ end portion of the first oligonucleotide primer annealed to the sample single-stranded nucleic acid molecule and continuing to at least the portion of the first single-stranded nucleic acid molecule which anneals with the second oligonucleotide primer, (ii) said displacing the second single-stranded nucleic acid molecule is carried out beginning from where the 3′ end portion of the second oligonucleotide primer annealed to the first single-stranded nucleic acid molecule and continuing to the end 3′ end of the second single-stranded nucleic acid molecule, or (iii) both (i) and (ii) are carried out.