Solid phase nucleic acid target capture and replication using strand displacing polymerases

Abstract

A method and kit for the capture and purification of specific nucleic acids from a sample with affinity capture probes on a solid support and for the replication of said nucleic acids with a strand displacing polymerase, whereby a second primer complementary to a sequence in each of the target nucleic acids distinct from that bound by capture probes is also bound to the nucleic acid targets, and extension of one of the primers on each target effects the separation of the copied nucleic acid strands from the solid support. Incorporation of universal nucleic acid sequences during their replication enables the simultaneous and highly specific amplification of multiple nucleic acid target sequences with minimal production of artifacts.

Therefore what is claimed is: 
     
         1 . A method for the purification and replication of target nucleic acids ( 1 ) from a sample, comprising:
 a) contacting said sample with reverse primer/capture probe oligonucleotides ( 2 ) having a 5′ tail comprising a sequence that can be used for amplification and incubating to anneal the reverse primer/capture probe oligonucleotides ( 2 ) that hybridize to target-specific sequences ( 17 ) on the target nucleic acids ( 1 ), whereby said incubating is performed in a buffer solution and not on a solid support ( 4 );   b) contacting said sample with reverse strand displacement primer oligonucleotides ( 3 ) and incubating to anneal the reverse strand displacement primer oligonucleotides ( 3 ) that hybridize to target-specific sequences ( 18 ) on the target nucleic acids ( 1 ) that are located on a 3′ side of the sequences ( 17 ) to which the reverse primer/capture probe oligonucleotides ( 2 ) hybridize to form soluble complexes of reverse primer/capture probe oligonucleotides ( 2 ) and reverse strand displacement primer oligonucleotides ( 3 ) bound to the target nucleic acids ( 1 ), whereby said incubating is performed in a buffer solution and not on the solid support ( 4 );   c) binding the formed soluble complexes previously formed in said incubating steps on a solid support ( 4 ) by binding a ligand on the reverse primer/capture probe oligonucleotides ( 2 ) to the solid support ( 4 ) and washing away excess reverse primer/capture probe oligonucleotides ( 2 ) and reverse strand displacement primer oligonucleotides ( 3 ) that are not bound to the target nucleic acids ( 1 ) and sample components other than the complexes;   d) after the step of washing away excess reverse primer/capture probe oligonucleotides ( 2 ) and reverse strand displacement primer oligonucleotides ( 3 ) and sample components other than the complexes, extending the reverse primer/capture probe oligonucleotides ( 2 ) with 5′ tail on the target nucleic acids ( 1 ) to form strands ( 5 ), and extending the reverse strand displacement primer oligonucleotides ( 3 ) on the target nucleic acids ( 1 ) with a nucleic acid strand displacing polymerase, wherein extension of the reverse strand displacement primer oligonucleotides ( 3 ) displaces, from the target nucleic acids ( 1 ), the extended reverse primer/capture probe oligonucleotides ( 2 ) to give single-stranded copies ( 5 ), which are copies of the target nucleic acids ( 1 ) and which are attached to the solid support ( 4 );   e) washing the double-stranded copies ( 6 ) resulting from the extension of reverse strand displacement primer oligonucleotides ( 3 ) that are not attached to the solid support ( 4 ) from the solid support ( 4 );   f) contacting a medium containing the single stranded copies bound to the solid support ( 4 ) with a solution containing a mixture of forward primers ( 7 ) with a 5′ tail comprising a sequence that can be used for amplification, that hybridize to target-specific sequences ( 20 ) on the single stranded copies ( 5 ), and forward strand displacement primers ( 8 ) that hybridize to target-specific sequences ( 19 ) on the single stranded copies ( 5 ), wherein the sequence ( 19 ) to which the forward strand displacement primers ( 8 ) hybridize is located on the 3′ side of the sequence ( 20 ) to which the forward primers ( 7 ) with a 5′ tail hybridize on the single stranded copy ( 5 ), and incubating to anneal forward primers ( 7 ) and forward strand displacement primers ( 8 ) to single stranded copies ( 5 ) to form complexes and washing away excess forward primers ( 7 ) and forward strand displacement primers ( 8 ) that are not bound to the single stranded copies ( 5 );   g) after washing away the excess forward primers ( 7 ) and forward strand displacement primers ( 8 ) extending forward primers ( 7 ) with tail 5′ and the forward strand displacement primers ( 8 ) hybridized on the single stranded copies ( 5 ) immobilized on the solid support ( 4 ) with a nucleic acid strand displacing polymerase, wherein extension of the strand displacement primers ( 8 ) displaces the single-stranded extension products ( 9 ) of the forward primers with 5′ tail ( 7 ) from the single stranded copies ( 5 ) and from the solid support ( 4 );   h) recovering the single-stranded extension products ( 9 ) in the supernatant solution bathing the solid support ( 4 ) said single-stranded extension products ( 9 ) containing the tail 5′ tail sequence of the forward primers ( 7 ) and the complement of the 5′ tail of the reverse primer/capture probe oligonucleotides ( 2 ), and adding them to with an amplification reagent mix; and   i) amplifying the extended forward primer strands ( 9 ) using one or both of the attached tail 5′ sequences.   
     
     
         2 . The method according to  claim 1 , wherein the reverse primer/capture probe oligonucleotides ( 2 ) that hybridize to target-specific sequences ( 17 ) on the target nucleic acids ( 1 ) and the reverse strand displacement primer oligonucleotides ( 3 ) that hybridize to target-specific sequences ( 18 ) on the target nucleic acids ( 1 ) in a position 3′ to the reverse primer/capture probe oligonucleotides ( 2 ) on the target nucleic acids ( 1 ) are hybridized simultaneously to the target nucleic acids ( 1 ). 
     
     
         3 . The method according to  claim 1 , wherein the reverse primer/capture probe oligonucleotides ( 2 ) that hybridize to target-specific sequences ( 17 ) on the target nucleic acids ( 1 ) and the reverse strand displacement primer oligonucleotides ( 3 ) that hybridize to target-specific sequences ( 18 ) on the target nucleic acids ( 1 ) in a position 3′ to the reverse primer/capture probe oligonucleotides ( 2 ) on the target nucleic acids ( 1 ) are hybridized separately to the target nucleic acids ( 1 ). 
     
     
         4 . The method according to  claim 1 , wherein amplification is effected with the polymerase chain reaction. 
     
     
         5 . The method according to  claim 1 , wherein amplification is effected with the strand displacement reaction. 
     
     
         6 . The method according to  claim 1 , wherein the tail sequences of the tail reverse primers ( 2 ) or the tailed forward primers ( 7 ) provide a promoter site for subsequent amplification. 
     
     
         7 . The method according to  claim 1 , wherein products of amplification are analyzed by hybridization to probes immobilized on a microarray. 
     
     
         8 . The method according to  claim 1 , wherein products of amplification are analyzed by determining a sequence of the amplification products. 
     
     
         9 . The method according to  claim 1 , wherein the tail sequences of the tail reverse primers ( 2 ) or the tailed forward primers ( 7 ) contain adapter sequences required for subsequent sequencing reactions or tags to identify specific samples or target nucleic acids ( 1 ). 
     
     
         10 . A kit comprising oligonucleotide primers and probes, nucleic acid polymerase enzymes, nucleotide triphosphates, solid supports ( 4 ), wash solutions and buffer solutions with divalent metal ions for performing any of the methods of  claim 1 . 
     
     
         11 . A method for the purification and replication of target nucleic acids ( 1 ) from a sample, comprising:
 a) contacting said sample with reverse strand displacement primer oligonucleotides ( 3 ) and incubating to anneal the reverse strand displacement primer oligonucleotides ( 3 ) to target-specific sequences ( 18 ) on the target nucleic acids ( 1 ) whereby said incubating is performed in a buffer solution and not on a solid support ( 4 );   b) contacting said sample with reverse primer/capture probe oligonucleotides ( 2 ) having a 5′ tail comprising a sequence that can be used for amplification and incubating to anneal the reverse primer/capture probe oligonucleotides ( 2 ) to target-specific sequences ( 17 ) on the target nucleic acids ( 1 ) that are located on the 5′ side of the sequences ( 18 ) to which the reverse strand displacement primer oligonucleotides ( 3 ) hybridize to form complexes of reverse primer/capture probe oligonucleotides ( 2 ) and reverse strand displacement primer oligonucleotides ( 3 ) bound to the target nucleic acids ( 1 );   c) purifying the formed complexes on a solid support ( 4 ) by capturing the complexes and washing away excess reverse primer/capture probe oligonucleotides ( 2 ) and reverse strand displacement primer oligonucleotides ( 3 ) that are not bound to the target nucleic acids ( 1 ) and sample components other than the complexes;   d) after the step of purifying said formed complexes, extending the reverse primer/capture probe oligonucleotides ( 2 ) with 5′ tail on the target nucleic acids ( 1 ) to form strands ( 5 ), and extending the reverse strand displacement primer oligonucleotides ( 3 ) on the target nucleic acids ( 1 ) with a nucleic acid strand displacing polymerase, wherein extension of the reverse strand displacement primer oligonucleotides ( 3 ) displaces, from the target nucleic acids ( 1 ), the extended reverse primer/capture probe oligonucleotides ( 2 ) to give single-stranded copies ( 5 ), which are copies of the target nucleic acids ( 1 ) and which are attached to the solid support ( 4 );   e) washing the double-stranded copies ( 6 ) resulting from the extension of reverse strand displacement primer oligonucleotides ( 3 ) that are not attached to the solid support ( 4 ) from the solid support ( 4 );   f) contacting a medium containing the single stranded copies ( 5 ) bound to the solid support ( 4 ) with solution containing a mixture of forward primers ( 7 ) with a 5′ tail comprising a sequence that can be used for amplification, that hybridize to target-specific sequences ( 20 ) on the single stranded copies ( 5 ), and forward strand displacement primers ( 8 ) that hybridize to target-specific sequences ( 19 ) on the single stranded copies ( 5 ), wherein the sequence ( 19 ) to which the forward strand displacement primers ( 8 ) hybridize is located on the 3′ side of the sequences ( 20 ) to which the forward primers ( 7 ) with a 5′ tail hybridize on the single stranded copies ( 5 ), and incubating to anneal forward primers ( 7 ) and forward strand displacement primers ( 8 ) to single stranded copies ( 5 ) to form complexes and washing away excess forward primers ( 7 ) and forward strand displacement primers ( 8 ) that are not bound to the single stranded copies ( 5 );   g) after washing away the excess forward primers ( 7 ) and forward strand displacement primers ( 8 ) extending forward primers ( 7 ) with tail 5′ and the forward strand displacement primers ( 8 ) hybridized on the single stranded copies ( 5 ) immobilized on the solid support ( 4 ) with a nucleic acid strand displacing polymerase, wherein extension of the strand displacement primers ( 8 ) displaces the single-stranded extension products ( 9 ) of the forward primers ( 7 ) with 5′ tail from the single stranded copies ( 5 ) and from the solid support ( 4 );   h) recovering the single-stranded extension products ( 9 ) in the supernatant solution bathing the solid support ( 4 ) said single-stranded extension products ( 9 ) containing the tail 5′ tail sequence of the forward primers ( 7 ) and the complement of the 5′ tail of the reverse primer/capture probe oligonucleotides ( 2 ), and adding them to with an amplification reagent mix; and   i) amplifying the extended forward primer strands ( 9 ) using one or both of the attached tail 5′ sequences.   
     
     
         12 . The method according to  claim 11 , wherein after step (a), steps (b) and (c) are preformed simultaneously. 
     
     
         13 . The method according to  claim 11 , wherein amplification is effected with the polymerase chain reaction. 
     
     
         14 . The method according to  claim 11 , wherein amplification is effected with the strand displacement reaction. 
     
     
         15 . The method according to  claim 11 , wherein the tail sequences of the tail reverse primers ( 2 ) or the tailed forward primers ( 7 ) provide a promoter site for subsequent amplification. 
     
     
         16 . The method according to  claim 11 , wherein products of amplification are analyzed by hybridization to probes immobilized on a microarray. 
     
     
         17 . The method according to  claim 11 , wherein products of amplification are analyzed by determining a sequence of the amplification products. 
     
     
         18 . The method according to  claim 11 , wherein the tail sequences of the tail reverse primers ( 2 ) or the tailed forward primers ( 7 ) contain adapter sequences required for subsequent sequencing reactions or tags to identify specific samples or target nucleic acids ( 1 ). 
     
     
         19 . A kit comprising oligonucleotide primers and probes, nucleic acid polymerase enzymes, nucleotide triphosphates, solid supports ( 4 ), wash solutions and buffer solutions with divalent metal ions for performing any of the methods of  claim 11 . 
     
     
         20 . A method for the purification and replication of target nucleic acids ( 1 ) from a sample, comprising:
 a) contacting said sample with reverse primer/capture probe oligonucleotides ( 2 ) having a 5′ tail comprising a sequence that can be used for amplification and incubating to anneal the reverse primer/capture probe oligonucleotides ( 2 ) to target-specific sequences ( 17 ) on the target nucleic acids ( 1 );   b) purifying said target nucleic acids ( 1 ) on a solid support ( 4 ) by capturing the complexes and washing away and sample components other than the complexes;   c) hybridizing reverse strand displacement primer oligonucleotides ( 3 ) to target-specific sequences ( 18 ) on the target nucleic acids ( 1 ) that are located on the 3′ side of the sequences ( 17 ) to which the reverse primer/capture probe oligonucleotides ( 2 ) hybridize;   d) washing the solid support ( 4 ) to remove excess strand displacement oligonucleotides ( 3 ) that are not bound to the target nucleic acids ( 1 );   e) extending the reverse primer/capture probe oligonucleotides ( 2 ) with 5′ tail on the target nucleic acids ( 1 ) to form strands ( 5 ), and extending the reverse strand displacement primer oligonucleotides ( 3 ) on the target nucleic acids ( 1 ) with a nucleic acid strand displacing polymerase, wherein extension of the reverse strand displacement primer oligonucleotides ( 3 ) displaces, from the target nucleic acids ( 1 ), the extended reverse primer/capture probe oligonucleotides ( 2 ) to give single-stranded copies ( 5 ), which are copies of the target nucleic acids ( 1 ) and which are attached to the solid support ( 4 );   f) washing the double-stranded copies ( 6 ) resulting from the extension of reverse strand displacement primer oligonucleotides ( 3 ) from the solid support ( 4 );   g) contacting a medium containing the single stranded copies ( 5 ) bound to the solid support ( 4 ) with solution containing a mixture of forward primers ( 7 ) with a 5′ tail comprising a sequence that can be used for amplification, that hybridize to target-specific sequences ( 20 ) on the single stranded copies ( 5 ), and forward strand displacement primers ( 8 ) that hybridize to target-specific sequences ( 19 ) on the single stranded copies ( 5 ), wherein the sequence ( 19 ) to which the forward strand displacement primers ( 8 ) hybridize is located on the 3′ side of the sequence ( 20 ) to which the forward primers ( 7 ) with a 5′ tail hybridize on the single stranded copies ( 5 ), and incubating to anneal forward primers ( 7 ) and forward strand displacement primers ( 8 ) to single stranded copies ( 5 ) to form complexes and washing away excess forward primers ( 7 ) and forward strand displacement primers ( 8 ) that are not bound to the single stranded copies ( 5 );   h) after washing away the excess forward primers ( 7 ) and forward strand displacement primers ( 8 ) extending forward primers ( 7 ) with tail 5′ and the forward strand displacement primers ( 8 ) hybridized on the single stranded copies ( 5 ) immobilized on the solid support ( 4 ) with a nucleic acid strand displacing polymerase, wherein extension of the strand displacement primers ( 8 ) displaces the single-stranded extension products ( 9 ) of the forward primers with 5′ tail ( 7 ) from the single stranded copies ( 5 ) and from the solid support ( 4 );   i) recovering the single-stranded extension products ( 9 ) in the supernatant solution bathing the solid support ( 4 ) said single-stranded extension products ( 9 ) containing the tail 5′ tail sequence of the forward primers ( 7 ) and the complement of the 5′ tail of the reverse primer/capture probe oligonucleotides ( 2 ), and adding them to with an amplification reagent mix; and   j) amplifying the extended forward primer strands ( 9 ) using one or both of the attached tail 5′ sequences.   
     
     
         21 . The method according to  claim 20 , wherein steps (a) and (b) are performed simultaneously. 
     
     
         22 . The method according to  claim 20 , wherein amplification is effected with the polymerase chain reaction. 
     
     
         23 . The method according to  claim 20 , wherein amplification is effected with the strand displacement reaction. 
     
     
         24 . The method according to  claim 20 , wherein the tail sequences of the tail reverse primers ( 2 ) or the tailed forward primers ( 7 ) provide a promoter site for subsequent amplification. 
     
     
         25 . The method according to  claim 20 , wherein products of amplification are analyzed by hybridization to probes immobilized on a microarray. 
     
     
         26 . The method according to  claim 20 , wherein products of amplification are analyzed by determining a sequence of the amplification products. 
     
     
         27 . The method according to  claim 20 , wherein the tail sequences of the tail reverse primers ( 2 ) or the tailed forward primers ( 7 ) contain adapter sequences required for subsequent sequencing reactions or tags to identify specific samples or target nucleic acids ( 1 ). 
     
     
         28 . A kit comprising oligonucleotide primers and probes, nucleic acid polymerase enzymes, nucleotide triphosphates, solid supports ( 4 ), wash solutions and buffer solutions with divalent metal ions for performing any of the methods of  claim 20 .