Methods for the multiplexed isothermal amplification of nucleic acid sequences
A method for the isothermal amplification of nucleic acid molecules, optionally on a solid support. The method uses single stranded nucleic acids having hairpin regions at both the 3′ and 5′ ends, or the extension products thereof.
1 . A method for the surface bound clonal concatameric amplification of a plurality of nucleic acid sequences, comprising:
(a) taking a population of different single-stranded nucleic acid molecules (ss1) of at least 50 nucleotides in length wherein the single strands have a different sequence and each of the single strands has a common hairpin region at the 3′ end (LP1) and a common hairpin region at the 5′ end (LP2), wherein each hairpin has 5′ and 3′ hybridising stem arms of between 15-30 base pairs in length, the stem arms having a Tm in the range of 45-55° C.; and
(b) clonally amplifying the population of different single-stranded nucleic acid molecules (ss1) using a reaction mixture containing a strand displacing polymerase, nucleotide monomers and amplification primers wherein at least one of the amplification primers (P1) is complementary to at least a portion of the loop of LP1 and to at least a portion of the 5′ stem arm of LP1 and one of the amplification primers (P2) hybridizes to at least a portion of a copy of the loop of LP2 (LP2′),
wherein in step (b) the amplification primer (P1) is attached to a solid support; and the population of single-stranded nucleic acid molecules are hybridized to the attached amplification primers (P1), such that single strands of different sequences are amplified in physical isolation; and the 3′ end of the stem arm of LP1 is extended to make a copy of the single-stranded nucleic acid molecules.
2 . The method according to claim 1 , wherein in step (b) the 3′ end of the stem arm of LP1 is extended to make a complete copy of the single-stranded nucleic acid molecules of at least 50 nucleotides in length (ss1′) and a complete copy of sequence LP2 (LP2′).
3 . The method according to claim 1 , wherein the amplification primer P2 is in solution.
4 . The method according to claim 1 , wherein the presence, absence or sequence of the nucleic acid amplification product is determined.