Compositions and methods for nucleic acid amplification

A kit comprising:

• a reaction mixture comprising:
• (a) more than one set of oligonucleotides, each set comprising a forward primer complementary to a strand of a target nucleic acid molecule and a reverse primer complementary to a complementary sequence of the strand of the target nucleic acid molecule:
• wherein the forward primer or the reverse primer of the set comprises a molecular moiety at 3′ end of said forward primer or said reverse primer, wherein the molecular moiety is non complementary to one or more nucleotides of the strand of the target nucleic acid molecule or said complement;
• (b) a polymerase with 3′-5′ exonuclease activity;
• and
• wherein the kit is configured to perform multiplex nucleic acid amplification of one or more target nucleic acid molecules such that an amplified product of the one or more target nucleic acid molecules is detectable by a detection device.

A method, comprising:

• (a) providing a reaction mixture comprising more than one set of oligonucleotides, each set comprising a forward primer complementary to a strand of a target nucleic acid molecule and a reverse primer complementary to a complementary sequence of the strand of the target nucleic acid molecule;
• wherein the forward primer or the reverse primer of the set comprises a molecular moiety at 3′ end of said forward primer or said reverse primer, wherein the molecular moiety is non complementary to one or more nucleotides of the strand of the target nucleic acid molecule or said complement;
• (b) annealing the forward primer to a strand of the target nucleic acid molecule, and the reverse primer to a complementary sequence to the strand of the target nucleic acid molecule,
• (c) removing the molecular moiety using a polymerase with 3′-5′ exonuclease activity;
• (d) extending the forward primer and the reverse primer in a template-directed manner to yield a double-stranded target nucleic acid molecule;
• (e) denaturing the double-stranded target nucleic acid molecule to generate single-stranded target nucleic acid molecules;
• and
• repeating (b)-(e) for at least one cycle to yield amplified double-stranded target nucleic acid molecules,
• wherein the method is configured to perform multiplex nucleic acid amplification of one or more target nucleic acid molecules.