Detection of differences in nucleic acids

A method of preparing a DNA partial duplex having a portion at an end thereof that has two predefined non-complementary single stranded sequences, said method comprising:

• (a) combining a medium containing a nucleic acid with a polymerase, nucleoside triphosphates and two primers, wherein one of said primers P3 is extendable along one of said strands of said nucleic acid, said P3 having a 3′-end portion Pa that binds to said one of said strands and a 5′-end portion A1 that does not bind to one of said strands, and the other of said primers P2 is extendable along the other of said strands of said nucleic acid, wherein the extended primer produced by the extension of one of said primers is a template for the other of said primers,
• (b) subjecting said medium to temperature cycling to extend said primers, and
• (c) combining said medium with a primer P1 wherein said P1 has said 3′-end portion Pa that binds to said extended primer P2 and a 5′-end portion B1 that does not bind to said extended P2 primer, and
• (d) subjecting said medium to conditions such that said P1, binds to and is extended along said extended primer P2 to produce only a complement.

A method of detecting a target nucleic acid sequence, said method comprising:

• (a) amplification of said target sequence by polymerase chain reaction, using primers P1 and P2 to produce an amplicon AA, wherein one of said primers P1 and P2 comprises a label and said primer P1 is comprised of a 3′-end portion Pa that can hybridize with said target sequence and 5′-end portion B1 that cannot hybridize with said target sequence,
• (b) extending a primer P3 by chain extension along one strand of amplicon AA to produce a tailed target partial duplex A′, wherein said primer P3 is comprised of said 3′-end portion Pa and a 5′-end portion A1 that cannot hybridize to said target sequence or its complement,
• (c) amplification of a reference nucleic acid sequence different than said target nucleic acid sequence, using said primer P2 and said primer P3, by polymerase chain reaction to produce amplicon BB, wherein said primer P2 comprises a label when said primer P2 in step (a) above comprises a label and said primer P3 comprises a label when said primer P1 in step (a) above comprises a label,
• (d) extending said primer P1 by chain extension along one strand of amplicon BB to produce a tailed reference partial duplex B′,
• (e) allowing said tailed target partial duplex A′ to bind to said tailed reference partial duplex B′ to form a complex, and
• (f) detecting the binding of one of said labels to another of said labels as a result of the formation of said complex, the binding thereof being directly related to the presence of said target nucleic acid sequence.

A method for detecting the presence of a difference between two related nucleic acid sequences, said method comprising:

• (a) producing from said two related nucleic acid sequences a double stranded target nucleic acid sequence and a double stranded reference nucleic acid sequence wherein each strand of said target nucleic acid sequence has a portion introduced therein that is a nucleotide sequence priming site and wherein each strand of said reference nucleic acid sequence has a portion introduced therein that is a nucleotide sequence priming site,
• (b) producing, from said target nucleic acid sequence using said nucleotide sequence priming sites, a partial duplex A′ comprising a fully complementary double stranded nucleic acid sequence containing said target nucleic acid sequence and a second portion wherein one strand of said double stranded sequence has at its 5′-end a portion A1, and the other strand has at its 3′ end a portion A2 that does not hybridize with portion A1,
• (c) producing, from said reference nucleic acid sequence using said nucleotide sequence priming sites, a partial duplex B′ comprising said double stranded nucleic acid reference sequence wherein the strand of B′ corresponding to the strand of A′ comprising said portion A1 has at its 5′-end a portion B1 that is complementary with said A2 and the other strand of said B′ has at its 3′-end a portion B2 that is complementary with said A1,
• (d) subjecting said partial duplexes A′ and B′ to conditions that permit said duplexes to hybridize to each other wherein, if said related nucleic acid sequences have a difference, a stable complex is formed comprising said partial duplex A′ and said partial duplex B′, and
• (e) determining whether said stable complex is formed, the presence thereof indicating the presence of said difference between said two related nucleic acid sequences.

A method for detecting the presence of a mutation in a target nucleic acid sequence, said method comprising:

• (a) amplification of said target nucleic acid sequence by polymerase chain reaction using primers PX1i and PX2i to produce a target sequence comprising nucleotide sequence priming sites Pa′ and P2′,
• (b) amplification of a reference nucleic acid sequence by polymerase chain reaction using primers PX1i and PX2i to produce a reference sequence comprising nucleotide sequence priming sites Pa′ and P2′, said reference sequence being identical to said target sequence but lacking a possible mutation,
• (c) amplification of said target sequence produced in step (a) by polymerase chain reaction, using primers P1 and P2 to produce an amplicon AA, wherein said primer P1 is comprised of a 3′-end portion Pa that can hybridize with priming site Pa′ of said target sequence and 5′-end portion B1 that cannot hybridize with said target sequence,
• (d) extending a primer P3 by chain extension along one strand of amplicon AA to produce a tailed target partial duplex A′, wherein said primer P3 is comprised of said 3′-end portion Pa and a 5′-end portion A1 that cannot hybridize to said target sequence or its complement,
• (e) amplification of said reference sequence produced in step (b), using said primer P2 and said primer P3, by polymerase chain reaction to produce amplicon BB,
• (f) extending said primer P1 by chain extension along one strand of amplicon BB to produce a tailed reference partial duplex B′,
• (g) allowing said tailed target partial duplex A′ to bind to said tailed reference partial duplex B′, and
• (h) detecting the formation of a complex between said tailed partial duplexes, the binding thereof being directly related to the presence of said mutation.

A kit for detecting a mutation in a target nucleic acid, said kit comprising in packaged combination:

• (a) a primer P2 that is extendable along one of said strands of said target nucleic acid,
• (b) a primer P1 comprising a 3′-end portion Pa that binds to, and is extendable along, the other of said strands of said target nucleic acid and a 5′-end portion B1 that does not bind to said target nucleic acid,
• (c) a primer P3 comprising said 3′-end portion Pa and a portion A1 that is different than said B1 and does not bind to said target nucleic acid, and
• (d) a pair of primers for amplifying said target and said reference nucleic acids wherein one of said primers has a 3′-end portion that is hybridizable to said target and said reference nucleic acids and a portion 5′ thereof that is not hybridizable with said target or said reference nucleic acids and is substantially identical to said primer P2 and the other of said primers has a 3′-end portion that is hybridizable to said target and said reference nucleic acids and a portion 5′ thereof that is not hybridizable with said target or said reference nucleic acids and is substantially identical to said 3′-end portion Pa of said primers P1 and P3.