Methods for true isothermal strand displacement amplification

A method for detecting an amplified target sequence using isothermal strand displacement amplification, the method comprising:

• (a) analyzing a double-stranded target nucleic acid sequence to locate high dissociation sequence regions, wherein the high dissociation sequence regions have an estimated fraction of dissociated bases of about 0.04 to about 0.2, and wherein the double-stranded target nucleic acid sequence lacks a natural nicking enzyme recognition site;
• (b) designing a forward primer and a reverse primer to hybridize to the high dissociation sequence regions of the double-stranded target nucleic acid sequence;
• (c) contacting a nucleic acid sample having the double-stranded target nucleic acid sequence with an amplification reaction mixture comprising:
• the forward primer and the reverse primer,
• wherein the forward primer has the formula:
• A-B,
• wherein B comprises a portion of the forward primer that is complementary to the target nucleic acid sequence, and wherein A comprises a portion of the forward primer that is non-complementary to the target nucleic acid sequence and comprises a forward nicking enzyme recognition sequence,
• wherein the reverse primer has the formula:
• A′-B′,
• wherein B′ comprises a portion of the reverse primer that is complementary to the target nucleic acid sequence, and wherein A′ comprises a portion of the reverse primer that is non-complementary to the target nucleic acid sequence and comprises a reverse nicking enzyme recognition sequence,
• a polymerase enzyme having strand displacement activity, and
• a nicking enzyme specific for the forward nicking enzyme recognition sequence or the reverse nicking enzyme recognition sequence;
• (b) incubating the amplification reaction mixture and the nucleic acid sample under amplification conditions suitable for amplification of a target nucleic acid to produce an amplified target nucleic acid, wherein the amplified nucleic acid comprises either a forward primer portion that is complementary to B or a reverse primer portion that is complementary to B′,
• wherein the contacting step and the incubating step are carried out at a temperature between about 40° C. and about 65° C. and amplification of the target nucleic acid occurs without a thermal denaturation step prior to amplification;
• and
• (c) detecting the amplified target nucleic acid by hybridizing an oligonucleotide probe to a detection portion of the amplified target nucleic acid, wherein the detection portion of the amplified target nucleic acid is non-complementary to and does not overlap with the forward primer portion or the reverse primer portion, and wherein the oligonucleotide probe comprises a minor groove binder (MGB), a fluorophore, and a quencher.