Methods for asymmetric semi-nested isothermal nucleotide amplification

A system for producing single-stranded oligonucleotide amplicons comprising asymmetric semi-nested isothermal nucleotide amplification of a target region of a template nucleic acid molecule, said system comprising:

• i) a sample comprising the template nucleic acid molecule;
• ii) a set of primers comprising:
• A) a first primer (P1) comprising a first template nucleotide sequence, wherein the entire first template nucleotide sequence is complementary to a first complementary binding region (CSBR1), wherein said P1 is 5′ to the target region, and wherein said CSBR1 is on a strand opposite to the target region;
• B) a second primer (P2) comprising a second template nucleotide sequence, wherein the entire second template nucleotide sequence is complementary to a second complementary binding region (CSBR2), wherein P2 is (i) 5′ to the target region or (ii) 5′ to the target region and comprises a portion of the target region, wherein said CSBR2 is on a strand opposite to the target region, and wherein P1 is at least partially 5′ to P2;
• and
• C) a third primer (P3) comprising a third template nucleotide sequence, wherein the entire third template nucleotide sequence is complementary to a target strand binding region (TSBR) of the target strand, wherein the TSBR comprises (i) at least a portion of the target region, (ii) a portion 3′ to the target region, or (iii) any combination of (i) and (ii), wherein the ratio of P1:P2:P3 in the set of primers is (1-10):(10-200):(1-20);
• and
• iii) a solution comprising one or more enzymes, one or more dNTPs, and a buffer, wherein the buffer comprises one or more buffering reagents, one or more salts, and one or more crowding reagents.