Detection of differences in nucleic acids

Abstract

A method is disclosed for detecting the presence of a difference between two related nucleic acid sequences. In the method a complex is formed comprising both strands of each sequence. Each member of at least one pair of non-complementary strands within the complex have labels. The association of the labels as part of the complex is determined as an indication of the presence of a difference between the two related sequences. The complex generally comprises a Holliday junction. In one aspect a medium suspected of containing said two related nucleic acid sequences is treated to provide partial duplexes having non-complementary tailed portions at one end. The double stranded portions of the partial duplexes are identical except for said difference. One of the strands of one of the partial duplexes is complementary to one of the strands of the other of the partial duplexes and the other of the strands of one of the partial duplexes is complementary to the other of the strands of the other of the partial duplexes. The medium is subjected to conditions that permit the binding of the tailed portions of the partial duplexes to each other. If there is a difference in the related nucleic acid sequences, a stable complex is formed comprising a Holliday junction. If no difference exists, the complex dissociates into duplexes. A determination is made whether the stable complex is formed, the presence thereof indicating the presence of the related nucleic acid sequences. The method has application in detecting the presence of a mutation in a target sequence or in detecting the target sequence itself.

What is claimed is:  
     
         1 . A method for detecting the presence of a difference between two related nucleic acid sequences, said method comprising: 
 (a) forming a complex comprising both of said nucleic acid sequences in double stranded form, wherein each member of at least one pair of non-complementary strands within said complex has a label and    (b) detecting the association of said labels within said complex, the association thereof being related to the presence of said difference.    
     
     
         2 . The method of  claim 1  wherein said difference is a mutation.  
     
     
         3 . The method of  claim 1  wherein said nucleic acid sequences are DNA.  
     
     
         4 . The method of  claim 1  wherein said complex comprises a Holliday junction.  
     
     
         5 . A method for detecting a mutation within a target nucleic acid sequence, said method comprising: 
 (a) forming from said target sequence a tailed target partial duplex A′ comprised of a duplex of said target sequence, a label and at one end of said duplex, two non-complementary oligonucleotides, one linked to each strand,    (b) providing in combination said tailed target partial duplex A′ and a tailed reference partial duplex B′ lacking said mutation having a label as a part thereof, wherein said tailed reference partial duplex B′ is comprised of two nucleic acid strands that are complementary to the strands in said tailed target partial duplex A′ but for the possible presence of a mutation and wherein said labels are present in non-complementary strands of said tailed target and tailed reference partial duplexes respectively, and    (c) detecting, by means of said labels, the formation of a complex between said tailed partial duplexes, the formation thereof being directly related to the presence of said mutation.    
     
     
         6 . The method of  claim 5  wherein said target nucleic acid sequence is DNA.  
     
     
         7 . The method of  claim 5  wherein said tailed reference partial duplex B′ is provided in said combination by forming said tailed reference partial duplex B′ in the same reaction medium as that used for step (a).  
     
     
         8 . The method of  claim 7  wherein forming said tailed target partial duplex A′ and said tailed reference partial duplex B′ is carried out simultaneously.  
     
     
         9 . The method of  claim 5  wherein said labels are independently selected from the group consisting of oligonucleotides, enzymes, dyes, fluorescent molecules, chemiluminescers, coenzymes, enzyme substrates, radioactive groups, small organic molecules and solid surfaces.  
     
     
         10 . The method of  claim 6  wherein said non-complementary oligonucleotides each have from 15 to 60 nucleotides.  
     
     
         11 . A method of detecting a mutation within a target nucleic acid sequence, said method comprising: 
 (a) amplification of said target sequence by polymerase chain reaction, using primers P 1  and P 2  to produce an amplicon AA, wherein one of said primers P 1  and P 2  comprises a label and wherein said primer P 1  is comprised of a 3′-end portion Pa that can hybridize with said target sequence and 5′-end portion B 1  that cannot hybridize with said target sequence,    (b) extending a primer P 3  by chain extension along one strand of amplicon AA to produce a tailed target partial duplex A′, wherein said primer P 3  is comprised of said 3′-end portion Pa and a 5′-end portion A 1  that cannot hybridize to said target sequence or its complement,    (c) amplification of a reference nucleic acid sequence, using said primer P 2  and said primer P 3 , by polymerase chain reaction to produce amplicon BB, said reference sequence being identical to said target sequence but lacking a possible mutation, wherein said primer P 2  comprises a label when said primer P 2  in step (a) above comprises a label and said primer P 3  comprises a label when said primer P 1  in step (a) above comprises a label,    (d) extending said primer P 1  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′, and    (f) detecting the binding of one of said labels to another of said labels as a result of the formation of a complex between said tailed partial duplexes, the binding thereof being directly related to the presence of said mutation.    
     
     
         12 . The method of  claim 11  wherein said amplification of step (c) is carried out in the same reaction medium as that used for step (a).  
     
     
         13 . The method of  claim 12  wherein said amplification of step (c) is carried out simultaneously with the amplification of step (a).  
     
     
         14 . The method of  claim 11  wherein the label of primer P 2  in step (c) is different than the label of primer P 2  in step (a).  
     
     
         15 . The method of  claim 14  wherein said labels are independently selected from the group consisting of oligonucleotides, enzymes, dyes, fluorescent molecules, chemiluminescers, coenzymes, enzyme substrates, radioactive groups, small organic molecules and solid surfaces.  
     
     
         16 . The method of  claim 14  wherein said nucleic acid is DNA.  
     
     
         17 . A method for detecting a mutation in a nucleic acid, said method comprising: 
 (a) producing, from a target nucleic acid sequence suspected of having a mutation, a partial duplex A′ comprising a fully complementary double stranded nucleic acid sequence containing said target nucleic acid sequence wherein one strand has at its 5′-end a portion A 1  that does not hybridize with a corresponding portion A 2  at the 3′-end of the other strand, wherein one of said strands of said partial duplex A′ comprises a label,    (b) producing, from a reference nucleic acid sequence that corresponds to said target nucleic acid sequence of step (a) except for said mutation, a partial duplex B′ comprising said double stranded nucleic acid sequence lacking said mutation wherein the strand corresponding to the strand comprising said portion A 1  has at its 5′-end a portion B 1  that is complementary with said A 2  and the other strand has at its 3′-end a portion B 2  that is complementary with said A 1 , wherein one of said strands of said partial duplex B′ comprises a label, said strand comprising said label being unable to hybridize directly to said strand of said partial duplex A′ that comprises a label,    (c) subjecting said partial duplexes A′ and B′ to conditions that permit said duplexes to hybridize to each other wherein, if said target nucleic acid sequence having said mutation is present, a stable complex is formed comprising said partial duplex A′ and said partial duplex B′, and    (d) determining whether said stable complex is formed, the presence thereof indicating the presence of said nucleic acid having said mutation.    
     
     
         18 . The method of  claim 17  wherein said labels are independently selected from the group consisting of oligonucleotides, enzymes, dyes, fluorescent molecules, chemiluminescers, coenzymes, enzyme substrates, radioactive groups, small organic molecules, polynucleotide sequences and solid surfaces.  
     
     
         19 . The method of  claim 17  wherein steps (a) and (b) are carried out simultaneously in the same reaction medium.  
     
     
         20 . The method of  claim 17  wherein said A 1  and said A 2  each have from 15 to 60 nucleotides.  
     
     
         21 . The method of  claim 17  wherein said nucleic acid is DNA.  
     
     
         22 . A method for detecting the presence of a difference between two related nucleic acid sequences, said method comprising: 
 (a) treating a medium suspected of containing said two related nucleic acid sequences to provide two partial duplexes each comprised of duplexes having at one end therein non-complementary end portions thereby forming two partial duplexes, wherein said partial duplexes are related in that, except for said difference, one of the strands S 1  of one of said partial duplexes is complementary to one of the strands S 1 ′ of the other of said partial duplexes and the other of the strands S 2  of said one of said partial duplexes is complementary to the other of the strands S 2 ′ of said other of said partial duplexes,    (b) subjecting said medium to conditions that permit the binding of S 1  to S 1 ′ and S 2  to S 2 ′, respectively, wherein, if there is a difference between said related nucleic acid sequences, a stable complex is formed comprising said strands S 1 , S 1 ′, S 2  and S 2 ′, and    (c) determining whether said stable complex is formed, the presence thereof indicating the presence of a difference between said related nucleic acid sequences.    
     
     
         23 . The method of  claim 22  wherein non-complementary strands within said complex have labels.  
     
     
         24 . The method of  claim 23  wherein the association of said labels as part of said complex is detected, the association thereof being related to the presence of said difference.  
     
     
         25 . The method of  claim 23  wherein said labels are independently selected from the group consisting of oligonucleotides, enzymes, dyes, fluorescent molecules, chemiluminescers, coenzymes, enzyme substrates, radioactive groups, small organic molecules, polynucleotide sequences and solid surfaces.  
     
     
         26 . 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 P 3  is extendable along one of said strands of said nucleic acid, said P 3  having a 3′-end portion Pa that does bind and a 5′-end portion A 1  that does not bind thereto, and the other of said primers P 2  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 P 1  wherein said P 1  has said 3′-end portion Pa and a 5′-end portion B 1  that does not bind to said extended P 2  primer, and    (d) subjecting said medium to conditions such that said P 1 , binds to and is extended along said extended primer P 2  to produce only a complement, and not a copy, of said extended primer.    
     
     
         27 . The method of  claim 26  wherein a label is bound to P 2  or P 1 .  
     
     
         28 . The method of  claim 27  wherein said labels are independently selected from the group consisting of oligonucleotides, enzymes, dyes, fluorescent molecules, chemiluminescers, coenzymes, enzyme substrates, radioactive groups, small organic molecules, polynucleotide sequences and solid surfaces.  
     
     
         29 . A method of preparing a DNA partial duplex having a portion at one end that has two non-complementary single stranded sequences, said method comprising: 
 (a) combining a medium containing a single stranded polynucleotide with a primer P 1  wherein said P 1  has a 3′-end portion Pa that binds to a sequence that is 8 to 60 nucleotides from the 3′-end of said single stranded polynucleotide and a 8 to 60 nucleotide portion B 1  that does not bind to said single stranded polynucleotide, and    (b) subjecting said medium to conditions under which P 1 , binds to and is extended along said single stranded polynucleotide.    
     
     
         30 . The method of  claim 29  wherein said P 1  comprises a label.  
     
     
         31 . The method of  claim 30  wherein said labels are independently selected from the group consisting of oligonucleotides, enzymes, dyes, fluorescent molecules, chemiluminescers, coenzymes, enzyme substrates, radioactive groups, small organic molecules, polynucleotide sequences and solid surfaces.  
     
     
         32 . A kit for detecting a mutation in a target nucleic acid, said kit comprising in packaged combination: 
 (a) a primer P 2  that is extendable along one of said strands of said target nucleic acid,    (b) a primer P 1  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 B 1  that does not bind to said target nucleic acid, and    (c) a primer P 3  comprising said 3′-end portion Pa and a portion A 1  that is different than said B 1  and does not bind to said target nucleic acid.    
     
     
         33 . The kit of  claim 32  which comprises a reference nucleic acid.  
     
     
         34 . The kit of  claim 32  which comprises: 
 (a) a polymerase,  
 (b) nucleoside triphosphates, and  
 (c) a pair of primers for amplifying said target and said reference nucleic acids.  
 
     
     
         35 . The kit of  claim 34  wherein all of said components are packaged in the same container.  
     
     
         36 . The kit of  claim 32  wherein at least one of said primers comprises a label.  
     
     
         37 . The kit of  claim 36  wherein said labels are independently selected from the group consisting of oligonucleotides, enzymes, dyes, fluorescent molecules, chemiluminescers, coenzymes, enzyme substrates, radioactive groups, small organic molecules, polynucleotide sequences and solid surfaces.  
     
     
         38 . A method for detecting a difference between two related nucleic acid sequences, said methods comprising: 
 (a) forming a quadramolecular complex comprising both of said nucleic acid sequences in double stranded form and    (b) detecting the presence of said complex by binding said complex to a receptor, the presence of said complex indicating the presence of a difference between said sequences.    
     
     
         39 . A method for detecting a target nucleic acid sequence, said method comprising: 
 (a) forming from said target sequence a tailed target partial duplex A′ comprised of a duplex of said target sequence, a label, and at one end of said duplex, two non-complementary oligonucleotides, one linked to each strand,    (b) providing in combination (i) said tailed target partial duplex A′ and (ii) a tailed reference partial duplex B′ comprising a duplex of a sequence different than said target sequence, a label and, at one end of said duplex, two oligonucleotides that are complementary to said two non-complementary oligonucleotides, one linked to each strand wherein said labels are on non-complementary strands, and    (c) detecting, by means of said labels, the formation of a complex between said partial duplexes A′ and B′, the formation thereof being directly related to the presence of said target nucleic acid.    
     
     
         40 . The method of  claim 39  wherein said target and said reference nucleic acid sequences are identical but for a mutation.  
     
     
         41 . The method of  claim 39  for detecting a target nucleic acid sequence that does not contain a mutation.  
     
     
         42 . A method of detecting a target nucleic acid sequence, said method comprising: 
 (a) amplification of said target sequence by polymerase chain reaction, using primers P 1  and P 2  to produce an amplicon AA, wherein one of said primers P 1  and P 2  comprises a label and said primer P 1  is comprised of a 3′-end portion Pa that can hybridize with said target sequence and 5′-end portion B 1  that cannot hybridize with said target sequence,    (b) extending a primer P 3  by chain extension along one strand of amplicon AA to produce a tailed target partial duplex A′, wherein said primer P 3  is comprised of said 3′-end portion Pa and a 5′-end portion A 1  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 P 2  and said primer P 3 , by polymerase chain reaction to produce amplicon BB, wherein said primer P 2  comprises a label when said primer P 2  in step (a) above comprises a label and said primer P 3  comprises a label when said primer P 1  in step (a) above comprises a label,    (d) extending said primer P 1  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.    
     
     
         43 . The method of  claim 42  wherein said target and said reference nucleic acid sequences are identical but for a mutation.  
     
     
         44 . The method of  claim 42  for detecting a target nucleic acid sequence that does not contain a mutation.  
     
     
         45 . A method for detecting a target nucleic acid sequence, said method comprising: 
 (a) producing, from a target nucleic acid sequence, a partial duplex A′ comprising a fully complementary double stranded nucleic acid sequence containing said target nucleic acid sequence wherein one strand has at its 5′-end a portion A 1  that does not hybridize with a corresponding portion A 2  at the 3′-end of the other strand, wherein one of said strands of said partial duplex A′ comprises a label,    (b) producing, from a reference nucleic acid sequence, a partiai duplex B′ comprising a double stranded nucleic acid sequence different from said target nucleic acid sequence, wherein the strand corresponding to the strand comprising said portion A 1  has at its 5′-end a portion B 1  that is complementary with said A 2  and the other strand has at its 3′-end a portion B 2  that is complementary with said A 1 , wherein one of said strands of said partial duplex B′ comprises a label, said strand comprising said label being unable to hybridize directly to said strand of said partial duplex that comprises a label,    (c) subjecting said partial duplexes A′ and B′ to conditions that permit said duplexes to hybridize to each other to form a quadramolecular complex, and    (d) determining whether said complex is formed, the presence thereof indicating the presence of said target nucleic acid sequence.    
     
     
         46 . The method of  claim 45  wherein said target and said reference nucleic acid sequences are identical but for a mutation.  
     
     
         47 . The method of  claim 45  for detecting a target nucleic acid sequence that does not contain a mutation.  
     
     
         48 . 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 target nucleic acid sequence and a reference nucleic acid sequence wherein each respective strand of said target nucleic acid sequence has a portion introduced therein that is a nucleotide sequence priming site and wherein each respective 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 wherein one strand has at its 5′-end a portion A 1  that does not hybridize with a corresponding portion A 2  at the 3′-end of the other strand,    (c) producing, from said reference nucleic acid sequence using said nucleotide sequence priming sites, a partial duplex B′ comprising said double stranded nucleic acid sequence wherein the strand corresponding to the strand comprising said portion A 1  has at its 5′-end a portion B 1  that is complementary with said A 2  and the other strand has at its 3′-end a portion B 2  that is complementary with said A 1 ,    (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.    
     
     
         49 . The method of  claim 48  wherein steps (a), (b) and (c) are carried by polymerase chain reaction and wherein said priming sites are introduced into each respective strand of said target nucleic acid sequence and said reference nucleic acid sequence in step (a) oligonucleotide primers each having a 3′-end portion that hybridizes to a respective strand of said related nucleic acid sequences and a portion 5′ of said 3′-end portion that does not hybridize to said strand and is capable of hybridizing to a respective oligonucleotide primer used in step (b) or step (c).  
     
     
         50 . The method of  claim 48  wherein one of the strands of said partial duplex A′ comprises a label and one of the strands of said partial duplex B′ comprises a label wherein said strand comprising said label is unable to hybridize directly to said strand of said partial duplex A′ that comprises a label.  
     
     
         51 . The method of  claim 50  wherein said labels are independently selected from the group consisting of oligonucleotides, enzymes, dyes, fluorescent molecules, chemiluminescers, coenzymes, enzyme substrates, radioactive groups, small organic molecules, polynucleotide sequences and solid surfaces.  
     
     
         52 . The method of  claim 48  wherein step (a) is carried out in a separate reaction container from that in which steps (b) and (c) are carried out.  
     
     
         53 . The method of  claim 48  wherein said A 1  and said A 2  each have from 15 to 60 nucleotides.  
     
     
         54 . The method of  claim 48  wherein said nucleic acid is DNA.  
     
     
         55 . 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 PX 1   i  and PX 2   i  to produce a target sequence comprising nucleotide sequence priming sites Pa′ and P 2 ′,    (b) amplification of a reference nucleic acid sequence by polymerase chain reaction using primers PX 1   i  and PX 2   i  to produce a reference sequence comprising nucleotide sequence priming sites Pa′ and P 2 ′, 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 P 1  and P 2  to produce an amplicon AA, wherein said primer P 1  is comprised of a 3′-end portion Pa that can hybridize with priming site Pa′ of said target sequence and 5′-end portion B 1  that cannot hybridize with said target sequence,    (d) extending a primer P 3  by chain extension along one strand of amplicon AA to produce a tailed target partial duplex A′, wherein said primer P 3  is comprised of said 3′-end portion Pa and a 5′-end portion A 1  that cannot hybridize to said target sequence or its complement,    (e) amplification of said reference sequence produced in step (b), using said primer P 2  and said primer P 3 , by polymerase chain reaction to produce amplicon BB,    (f) extending said primer P 1  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.    
     
     
         56 . The method of  claim 55  wherein said amplification of step (b) is carried out in the same reaction medium as that used for step (a) and in a different reaction medium from that used for steps (c) and (e).  
     
     
         57 . The method of  claim 55  wherein said amplification of step (b) is carried out simultaneously with the amplification of step (a).  
     
     
         58 . The method of  claim 55  wherein in step (c) one of said primers P 1  and P 2  comprises a label and wherein in step (e) said primer P 2  comprises a label when said primer P 2  in step (c) comprises a label and said primer P 3  comprises a label when said primer P 1  in step (c) comprises a label.  
     
     
         59 . The method of  claim 58  wherein the label of primer P 2  in step (e) is different than the label of primer P 2  in step (c).  
     
     
         60 . The method of  claim 59  wherein said labels are independently selected from the group consisting of oligonucleotides, enzymes, dyes, fluorescent molecules, chemiluminescers, coenzymes, enzyme substrates, radioactive groups, small organic molecules and solid surfaces.  
     
     
         61 . The method of  claim 59  wherein said nucleic acid is DNA.  
     
     
         62 . A kit for detecting a mutation in a target nucleic acid, said kit comprising in packaged combination: 
 (a) a primer P 2  that is extendable along one of said strands of said target nucleic acid,    (b) a primer P 1  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 B 1  that does not bind to said target nucleic acid,    (c) a primer P 3  comprising said 3′-end portion Pa and a portion A 1  that is different than said B 1  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 P 2  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 P 1  and P 3 .    
     
     
         63 . The kit of  claim 62  which comprises a reference nucleic acid.  
     
     
         64 . The kit of  claim 62  which comprises: 
 (a) a polymerase and  
 (b) nucleoside triphosphates.  
 
     
     
         65 . The kit of  claim 62  wherein components (a)-(c) are packaged in one container and components (d) are packaged in a separate container.  
     
     
         66 . The kit of  claim 62  wherein at least one of said primers P 1 , P 2  or P 3  comprises a label.  
     
     
         67 . The kit of  claim 66  wherein said labels are independently selected from the group consisting of oligonucleotides, enzymes dyes, fluorescent molecules, chemiluminescers, coenzymes, enzyme substrates, radioactive groups, small organic molecules, polynucleotide sequences and solid surfaces.