Method of detecting nucleic acid

Abstract

The present invention relates to a method of detecting or quantifying a target nucleic acid present in a specimen. The present invention provides a method of detecting or quantifying a target nucleic acid by using artificially-designed probes having a flag consisting of a plurality of units, thereby easily and accurately detecting the target nucleic acid.

What is claimed is:  
     
         1 . A method of detecting or quantifying a target nucleic acid having a predetermined sequence in a specimen comprising: 
 (a) preparing a probe A and a probe B,    said probe A being a first probe which has a sequence F′ complementary to a first partial sequence F of the target nucleic acid and a binding molecule bound to the sequence F′, and    said probe B being a second probe which has a sequence S′ complementary to a second partial sequence S of the target nucleic acid and a flag bound to the sequence S′, where said flag is a double-stranded sequence and has a marker substance in one of the double strand;    (b) hybridizing the first probe A with the first partial sequence F of the target nucleic acid and hybridizing the second probe B with the second partial sequence S of the target nucleic acid;    (c) ligating the first probe A and the second probe B both being hybridized with the target nucleic acid, thereby obtaining a probe (A+B);    (d) binding the binding molecule to a substance capable of being paired up therewith, thereby recovering the probe (A+B); and    (e) recovering a single-stranded nucleic acid having the marker substance of the double stranded nucleic acid constituting the flag and detecting or quantifying the marker substance, thereby detecting or quantifying the target nucleic acid in the specimen.    
     
     
         2 . A method of detecting or quantifying target nucleic acids N 1 -Nn (n is an integer of 2 or more), each having a predetermined sequence, in a specimen, comprising: 
 (a) preparing probes A 1 -An (n is an integer of 2 or more) and probes B 1 -Bn (n is an integer of 2 or more),    said probes A 1 -An being first probes which respectively have sequences F 1 ′-Fn′ (n is an integer of 2 or more) complementary to first partial sequences F 1 -Fn (n is an integer of 2 or more) of the target nucleic acids and a binding molecule bound to each of the sequences F 1 ′-Fn′, and    said probes B 1 -Bn (n is an integer of 2 or more) being second probes which respectively have sequences S 1 ′-Sn′ (n is an integer of 2 or more) complementary to second partial sequences S 1 -Sn (n is an integer of 2 or more) of the target nucleic acids and flags bound to the sequences S 1 ′-Sn′, where each of said flags is a double-stranded sequence and has a marker substance in one of the double strand; and    (b) respectively hybridizing the first probes A 1 -An with the first partial sequences F 1 -Fn of the target nucleic acids, and simultaneously hybridizing the second probes B 1 -Bn with the second partial sequences S 1 -Sn of the target nucleic acids, respectively;    (c) respectively ligating the first probes A 1 -An and the second probes B 1 -Bn, both being hybridized with the target nucleic acids, respectively, thereby obtaining probes (A 1 +B 1 )-(An+Bn) (n is an integer of 2 or more);    (d) binding the binding molecule to a substance capable of being paired up therewith, thereby recovering the probes (A 1 +B 1 )-(An+Bn); and    (e) recovering a single-stranded nucleic acid having the marker substance from the double-stranded nucleic acid constituting each of the flags and detecting or quantifying the marker substance, thereby detecting or quantifying each of the target nucleic acids N 1 -Nn in the specimen.    
     
     
         3 . A method of detecting or quantifying a target nucleic acid having a predetermined sequence, in a specimen, comprising: 
 (a) preparing a probe A and a probe B, said probe A being a first probe which has a sequence F′ complementary to a first partial sequence F of the target nucleic acid and a tag sequence Tg bound to the sequence F′, and    said probe B being a second probe which has a sequence S′ complementary to a second partial sequence S of the target nucleic acid and a marker substance bound to the sequence S′   (b) mixing the probe A, the probe B, and the specimen, thereby hybridizing the probe A with the first partial sequence F of the target nucleic acid and simultaneously hybridizing the probe B with the second partial sequence S of the target nucleic acid;    (c) ligating the probe A and the probe B, both being hybridized with the target nucleic acid, thereby obtaining a probe (A+B);    (d) dissociating the probe (A+B) from the target nucleic acid;    (e) hybridizing the tag sequence Tg with a sequence Tg 1  complementary to the tag sequence Tg, thereby recovering the probe (A+B); and    (f) detecting or quantifying the marker substance in the probe (A+B) recovered, thereby detecting or quantifying the target nucleic acid in the specimen.    
     
     
         4 . A method of detecting or quantifying nucleic acids N 1 -Nn, each having a predetermined sequence, in a specimen, comprising: 
 (a) preparing probes A 1 -An (n is an integer of 2 or more) and probes B 1 -Bn (n is an integer of 2 or more),    said probes A 1 -An being first probes which respectively have sequences F 1 ′-Fn′ (n is an integer of 2 or more) complementary to first partial sequences F 1 -Fn (n is an integer of 2 or more) of the target nucleic acids N 1 -Nn (n is an integer of 2 or more) and tag sequences Tg 1 -Tgn bound to the sequences F 1 ′-Fn′, and    said probes B 1 -Bn being second probes which respectively have sequences S 1 ′-Sn′ (n is an integer of 2 or more) complementary to second partial sequences S 1 -Sn (n is an integer of 2 or more) of the target nucleic acids N 1 -Nn and the marker substance bound to each of the sequences S 1 ′-Sn′ of the target nucleic acid;    (b) mixing the probes A 1 -An, the probes B 1 -Bn, and the specimen, thereby hybridizing probes A 1 -An respectively with the first partial sequences F 1 -Fn of the target nucleic acids N 1 -Nn, and simultaneously hybridizing the probes B 1 -Bn with the second partial sequences S 1 -Sn of the target nucleic acids N 1 -Nn, respectively;    (c) respectively ligating probes A 1 -An and the probes B 1 -Bn, both being hybridized with the target nucleic acids, respectively, thereby obtaining probes (A 1 +B 1 )-(An+Bn) (n is an integer of 2 or more);    (d) dissociating the probes (A 1 +B 1 )-(An+Bn) from the target nucleic acids;    (e) hybridizing sequences Tg 1 -Tgn respectively with sequences Tg 1 ′-Tgn′ complementary to the tag sequences Tg 1 -Tgn, thereby recovering the probes (A 1 +B 1 )-(An+Bn); and    (f) detecting or quantifying the marker substance in the probes (A 1 +B 1 )-(An+Bn) recovered, thereby detecting or quantifying the target nucleic acids N 1 -Nn in the specimen.    
     
     
         5 . A method of detecting or quantifying a target nucleic acid having a predetermined sequence in a specimen, comprising: 
 (a) preparing a probe A and a probe B,    said probe A being a first probe which has a sequence F′ complementary to a first partial sequence F of the target nucleic acid and a tag sequence Tg bound to the sequence F′, and    said probe B being a second probe which has a sequence S′ complementary to a second partial sequence S of the target nucleic acid, a flag sequence FL bound to the sequence S′, and a marker substance bound to the flag sequence FL;    (b) mixing the probe A, the probe B, and the specimen, thereby hybridizing the probe A with the first partial sequence F of the target nucleic acid and simultaneously hybridizing the probe B with the second partial sequence S of the target nucleic acid;    (c) ligating the probe A and the probe B, both being hybridized with the target nucleic acid, thereby obtaining a probe (A+B);    (d) dissociating the probe (A+B) from the target nucleic acid;    (e) hybridizing the tag sequence Tg contained in the probe (A+B) with a sequence Tg′ complementary to the tag sequence Tg, thereby dissociating the probe (A+B); and    (f) recovering a portion containing at least the probe B from the prove (A+B) hybridized with the sequence Tg 1 ;    (g) hybridizing the flag sequence FL recovered with a nucleic acid sequence FL′ complementary to the flag sequence FL, thereby specifically recovering the portion containing at least probe B; and    (h) selectively detecting the marker substance contained in the portion containing at least the probe B recovered, thereby detecting or quantifying the target nucleic acid in the specimen.    
     
     
         6 . A method of detecting or quantifying nucleic acids N 1 -Nn (n is an integer of 2 or more), each having a predetermined sequence, in a specimen, comprising: 
 (a) preparing probes A 1 -An (n is an integer of 2 or more) and probes B 1 -Bn (n is an integer of 2 or more),    said probes A 1 -An being first probes which respectively have sequences F 1 ′-Fn′ (n is an integer of 2 or more) complementary to first partial sequences F 1 -Fn of the target nucleic acids N 1 -Nn (n is an integer of 2 or more), respectively, and tag sequences Tg 1 -Tgn bound to the sequences F 1 ′-Fn′, respectively, and    said probes B 1 -Bn being second probes which respectively have sequences S 1 ′-Sn′ (n is an integer of 2 or more) complementary to second partial sequences S 1 -Sn (n is an integer of 2 or more) of the target nucleic acids N 1 -Nn, flag sequences FL 1 -FLn bound to the sequences S 1 ′-Sn′, and a marker substance bound to each of the flag sequences FL 1 ′-FLn′;    (b) mixing the probes A 1 -An, the probes B 1 -Bn, and the specimen, hybridizing probes A 1 -An respectively with the first partial sequences F 1 -Fn of the target nucleic acids N 1 -Nn, and simultaneously hybridizing the probes B 1 -Bn with the second partial sequences S 1 -Sn of the target nucleic acids N 1 -Nn, respectively;    (c) respectively ligating probes A 1 -An and second probes B 1 -Bn, both being hybridized with the target nucleic acids, thereby obtaining probes (A 1 +B 1 )-(An+Bn) (n is an integer of 2 or more);    (d) dissociating the probes (A 1 +B 1 )-(An+Bn) from the target nucleic acids;    (e) hybridizing tag sequences Tg 1 -Tgn contained in the probes (A 1 +B 1 )-(An+Bn) with sequences Tg 1 ′-Tgn′ complementary to the tag sequences Tg 1 -Tgn, thereby dissociating the probes (A 1 +B 1 )-(An+Bn); and    (f) recovering portions respectively containing at least the probes B 1 -Bn, from the probes (A 1 +B 1 )-(An+Bn) hybridized with the sequence Tg 1 ′-Tgn′;    (g) hybridizing the flag sequences FL 1 -FLn with nucleic acid sequences FL 1 -FLn′ complementary to the flag sequences FL 1 -FLn, thereby specifically recovering the portions respectively containing at least probes B 1 -Bn; and    (h) selectively detecting the marker substance contained in the portions respectively containing at least the probes B 1 -Bn recovered, thereby detecting or quantifying the target nucleic acids N 1 -Nn in the specimen.    
     
     
         7 . A method of detecting or quantifying nucleic acids N 1 -Nn (n is an integer of 2 or more), each having a predetermined sequence, in a specimen, comprising: 
 (a) preparing probes A 1 -An (n is an integer of 2 or more) and probes B 1 -Bn (n is an integer of 2 or more),    said probes A 1 -An being first probes which respectively have sequences F 1 ′-Fn′ (n is an integer of 2 or more) complementary to first partial sequences F 1 -Fn of the target nucleic acids N 1 -Nn (n is an integer of 2 or more) and tag sequences Tg 1 -Tgn bound to the sequences F 1 ′-Fn′, respectively, and said probes B 1 -Bn being second probes which respectively have sequences S 1 ′-Sn′ (n is an integer of 2 or more) complementary to second partial sequences S 1 -Sn (n is an integer of 2 or more) of the target nucleic acids N 1 -Nn, and a marker substance bound to each of the sequences S 1 ′-Sn′   (b) mixing the probes A 1 -An, the probes B 1 -Bn, and the specimen, thereby hybridizing probes A 1 -An respectively with the first partial sequences F 1 -Fn of the target nucleic acids N 1 -Nn, and simultaneously hybridizing the probes B 1 -Bn with the second partial sequences S 1 -Sn of the target nucleic acids N 1 -Nn, respectively;    (c) respectively ligating probes A 1 -An and second probes B 1 -Bn, both being hybridized with the target nucleic acids N 1 -Nn, thereby obtaining probes (A 1 +B 1 )-(An+Bn) (n is an integer of 2 or more);    (d) hybridizing tag sequences Tg 1 -Tgn with sequences Tg 1 ′-Tgn′ complementary to the tag sequences Tg 1 -Tgn, thereby recovering the probes (A 1 +B 1 )-(An+Bn); and    (e) detecting or quantifying the marker substance contained in the probes (A 1 +B 1 )-(An+Bn) recovered, thereby detecting or quantifying the target nucleic acids N 1 -Nn in the specimen, p 1  wherein Tm values of the tag sequences Tg 1 -Tgn are higher than Tm values of sequences F 1 -Fn and sequences S 1 -Sn.    
     
     
         8 . A method of detecting or quantifying a target nucleic acid having a predetermined sequence in a specimen comprising: 
 (a) preparing a probe A and a probe B,    said probe A being a first probe which has a sequence F′ complementary to a first partial sequence F of the target nucleic acid and a binding molecule bound to the sequence F′, and    said probe B being a second probe which has a sequence S′ complementary to a second partial sequence S of the target nucleic acid and a flag sequence FL consisting of 4 units bound to the sequence S′, where said flag FL sequence hybridizes with a sequence FL′ bound to the sequence S′ to form a double-stranded sequence; and    (b) mixing the probe A, probe B and the specimen, thereby hybridizing the probe A with the first partial sequence F of the target nucleic acid, and simultaneously hybridizing the second probe B with the second partial sequence S of the target nucleic acid;    (c) ligating the probe A and the probe B, both being hybridized with the target nucleic acid, thereby obtaining a probe (A+B);    (d) binding the binding molecule to a substance capable of being paired up therewith, thereby recovering the probe (A+B); and    (e) denaturing the double-stranded flag sequence of the probes (A+B) recovered into single-stranded flag sequence;    (f) hybridizing the single-stranded flag sequence with two primers one of which has a binding molecule B and the other of which has a marker substance L, and extending the primers to form a complementary strand of the flag sequence FL, thereby obtaining a double strand;    (g) binding a binding molecule B with a substance capable of being paired with the binding molecule B, thereby recovering the double strand; and    (h) detecting or quantifying the target substance L, thereby detecting or quantifying the target nucleic acid in the specimen.    
     
     
         9 . A method of detecting or quantifying target nucleic acids N 1 -Nn (n is an integer of 2 or more), each having a predetermined sequence, in a specimen, comprising: 
 (a) preparing probes A 1 -An (n is an integer of 2 or more) and probes B 1 -Bn (n is an integer of 2 or more),    said probes A 1 -An being first probes which respectively have sequences F 1 ′-Fn′ (n is an integer of 2 or more) complementary to first partial sequences F 1 -Fn (n is an integer of 2 or more) of the target nucleic acids and a binding molecule bound to each of the sequences F 1 ′-Fn′, and    said probes B 1 -Bn (n is an integer of 2 or more) being second probes which respectively have sequences S 1 ′-Sn′ (n is an integer of 2 or more) complementary to second partial sequences S 1 -Sn (n is an integer of 2 or more) of the target nucleic acids, and flag sequences FL 1 -FLn each consisting of 4 units, bound to the sequences S 1 ′-Sn′, where said flag sequences FL 1 -FLn hybridize respectively with sequences FL 1 ′-FLn′ bound to the sequences S 1 ′-Sn′ to form double-stranded sequences; and    (b) mixing the probes A 1 -An, the probes B 1 -Bn, and the specimen, thereby hybridizing probes A 1 -An respectively with the first partial sequences F 1 -Fn of the target nucleic acids N 1 -Nn, and simultaneously hybridizing the probes B 1 -Bn with the second partial sequences S 1 -Sn of the target nucleic acids N 1 -Nn;    (c) respectively ligating the probes A 1 -An and the probes B 1 -Bn, both being hybridized with the target nucleic acids N 1 -Nn, thereby obtaining probes (A 1 +B 1 )-(An+Bn);    (d) binding each of the binding molecules to a substance capable of being paired up therewith, thereby recovering the probes (A 1 +B 1 )-(An+Bn); and    (e) denaturing double-stranded flag sequences of the probes (A+B)-(An+Bn) recovered into single-stranded flag sequences;    (f) hybridizing the single-stranded flag sequences FL 1 -FLn with two primers one of which has a binding molecule B and the other of which has a marker substance L, and extending the two primers, to form complementary strands of the flag sequences FL 1 -FLn, thereby obtaining double strands;    (g) binding a binding molecule B with a substance capable of being paired therewith, thereby recovering the double strands; and    (h) detecting or quantifying the marker substance L, thereby detecting or quantifying the target nucleic acids N 1 -Nn in the specimen.    
     
     
         10 . A method of detecting or quantifying a target nucleic acid having a predetermined sequence in a specimen comprising: 
 (a) preparing a probe A and a probe B,    said probe A being a first probe which has a sequence F′ complementary to a first partial sequence F of the target nucleic acid and a binding molecule bound to the sequence F′, and    said probe B being a second probe which has a sequence S′ complementary to a second partial sequence S of the target nucleic acid and a flag consisting of 4 units bound to the sequence S′, where said flag FL is a double-stranded sequence; and    (b) mixing the probe A, the probe B, and the specimen, thereby hybridizing the probe A with the first partial sequence F of the target nucleic acid and simultaneously hybridizing the probe B with the second partial sequence S of the target nucleic acid;    (c) ligating the probe A and the probe B, both being hybridized with the target nucleic acid, thereby obtaining a probe (A+B);    (d) binding the binding molecule to a substance capable of being paired up therewith, thereby recovering the probe (A+B); and    (e) denaturing the double-stranded nucleic acid constituting the flag into single-stranded nucleic acid;    (f) amplifying the single-stranded nucleic acid present in a liquid phase by PCR, thereby performing an encode reaction;    (g) performing transcription of a sequence FL′ complementary to the single stranded flag sequence obtained by the encode reaction, by use of two primers one of which is a primer having another binding molecule and the other of which is a primer having a marker substance, thereby performing a decode reaction;    (h) binding said another binding molecule to a substance being paired up therewith, recovering a nucleic acid molecule obtained by the decode reaction; and    (i) detecting or quantifying the marker substance, thereby detecting or quantifying the target nucleic acid.    
     
     
         11 . A method of detecting or quantifying target nucleic acids N 1 -Nn (n is an integer of 2 or more), each having a predetermined sequence, in a specimen, comprising: 
 (a) preparing probes A 1 -An (n is an integer of 2 or more) and probes B 1 -Bn (n is an integer of 2 or more),    said probes A 1 -An being first probes which respectively have sequences F 1 ′-Fn′ (n is an integer of 2 or more) complementary to first partial sequences F 1 -Fn (n is an integer of 2 or more) of the target nucleic acids and a binding molecule bound to each of the sequences F 1 ′-Fn′, and    said probes B 1 -Bn (n is an integer of 2 or more) being second probes which respectively have sequences S 1 ′-Sn′ (n is an integer of 2 or more) complementary to second partial sequences S 1 -Sn (n is an integer of 2 or more) of the target nucleic acids and flag sequences FL 1 -FLn each consisting of 4 units, bound to the sequences S 1 ′-Sn′;    (b) mixing the first probes A 1 -An, the second probes B 1 -Bn, and the specimen, thereby hybridizing the probes A 1 -An respectively with the first partial sequences F 1 -Fn of the target nucleic acids N 1 -Nn and simultaneously hybridizing the probes B 1 -Bn with the second partial sequences S 1 -Sn of the target nucleic acids N 1 -Nn, respectively;    (c) respectively ligating the probes A 1 -An and the probes B 1 -Bn, both being hybridized with the target nucleic acids N 1 -Nn, thereby obtaining probes (A 1 +B 1 )-(An+Bn) (n is an integer of 2 or more);    (d) binding the binding molecule to a substance capable of being paired up therewith, to recover the probes (A 1 +B 1 )-(An+Bn), and thereafter performing an encode reaction of each of the flags FL 1 -FLn; and    (e) performing a decode reaction of the sequences FL 1 ′-FLn′ complementary to the flags FL 1 -FLn obtained by the encode reaction; and    (f) detecting or quantifying the nucleic acid molecules obtained by the decode reaction, thereby detecting or quantifying the target nucleic acids N 1 -Nn in the specimen.    
     
     
         12 . A method of detecting or quantifying target nucleic acids N 1 -Nn (n is an integer of 2 or more), each having a predetermined sequence, in a specimen, comprising: 
 (a) preparing probes A 1 -An (n is an integer of 2 or more) and probes B 1 -Bn (n is an integer of 2 or more),    said probes A 1 -An being first probes which respectively have sequences F 1 ′-Fn′ (n is an integer of 2 or more) complementary to first partial sequences F 1 -Fn (n is an integer of 2 or more) of the target nucleic acids and a binding molecule bound to each of the sequences F 1 ′-Fn′, and    said probes B 1 -Bn (n is an integer of 2 or more) being second probes which respectively have sequences S 1 ′-Sn′ (n is an integer of 2 or more) complementary to second partial sequences S 1 -Sn (n is an integer of 2 or more) of the target nucleic acids and flag sequences FL 1 -FLn each consisting of 4 units, bound to the sequences S 1 ′-Sn′, respectively,    (b) mixing the probes A 1 -An, the probes B 1 -Bn, and the specimen, thereby hybridizing probes A 1 -An respectively with the first partial sequences F 1 -Fn of the target nucleic acids N 1 -Nn, and simultaneously hybridizing the probes B 1 -Bn with the second partial sequences S 1 -Sn of the target nucleic acids N 1 -Nn, respectively;    (c) respectively ligating the probes A 1 -An and the probes B 1 -Bn, both being hybridized with the target nucleic acids N 1 -Nn, thereby obtaining probes (A 1 +B 1 )-(An+Bn);    (d) binding each of the binding molecules to a substance capable of being paired up therewith to recover the probes (A 1 +B 1 )-(An+Bn), and thereafter performing an encode reaction for each of the flags FL 1 -FLn; and    (e) performing a decode reaction of the sequences Fl 1 ′-FLn′ complementary to the flags FL 1 -FLn (n is an integer of 2 or more) obtained by the encode reaction; and    (h) detecting the nucleic acid molecules obtained by the decode reaction, thereby detecting or quantifying the target nucleic acids N 1 -Nn in the specimen,    wherein 2 units of 4 units are sequences functioning as primers for PCR amplification.    
     
     
         13 . A method of detecting or quantifying a target nucleic acid having a predetermined sequence in a specimen comprising: 
 (a) preparing a probe A and a probe B, said probe A being a first probe which has a sequence F′ complementary to a first partial sequence F of the target nucleic acid and a binding molecule bound to the sequence F′, and    said probe B being a second probe which has a sequence S′ complementary to a second partial sequence S of the target nucleic acid and a flag consisting of 4 units bound to the sequence S′, where said flag FL is a double-stranded sequence and said 4 units consist of SD, D 0 , D 1  and ED each having an arbitrary sequence, bounded to each other sequentially in the order mentioned; and    (b) mixing the probe A, the probe B, and the specimen, thereby hybridizing the probe A with the first partial sequence F of the target nucleic acid and simultaneously hybridizing the probe B with the second partial sequence S of the target nucleic acid;    (c) ligating the probe A and the probe B both being hybridized with the target nucleic acid, thereby obtaining a probe (A+B);    (d) binding the binding molecule to a substance capable of being paired up therewith, thereby recovering the probe (A+B); and    (e) denaturing the double-stranded nucleic acid constituting the flag into a single-stranded nucleic acid;    (f) hybridizing the single-stranded nucleic acid obtained in a liquid phase with sequences complementary to sequences D 11 -D 1 n labeled with a marker substance, as primers,    (g) extending the primers hybridized    (h) denaturing a double-stranded nucleic acid having primers into a single-stranded nucleic acid;    (i) hybridizing the sequences D 01 -D 0 n specifically with the primers extended to detect or quantify the marker substances included in the sequences D 01 -D 0 n, thereby detecting or quantifying the target nucleic acids.    
     
     
         14 . The method according to  claims 10  to  12 , wherein the decode reaction comprises, where said flag(s) FL is a double-stranded sequence and said 4 units consist of SD, D 0 , D 1  and ED each having an arbitrary sequence, bound to each other sequentially in the order mentioned, 
 (i) performing PCR for a single-stranded sequence encoded using SD sequence to which a binding molecule is attached, and ED sequence, as primers;  
 (ii) binding a binding molecule bound to the SD sequence to a substance capable of being paired up therewith, thereby recovering a PCR product;  
 (iii) denaturing the PCR produce into a single strand  
 (iv) hybridizing the single strand with primers D 11 ′-D 1 n′ labeled;  
 (v) extending the primers;  
 (vi) denaturing the primers extended into single strands;  
 (vii) hybridizing extended single strands of the primers with sequences D 01 -D 0 n to detect or quantify marker substances included in that sequences D 01 -D 0 n, thereby detecting or quantifying the target nucleic acid.  
 
     
     
         15 . The method according to  claims 10  to  12 , wherein the decode reaction comprises, where said flag FL is a double-stranded sequence and said 4 units consist of SD, D 0 , D 1  and ED each having an arbitrary sequence, bound to each other sequentially in the order mentioned; and 
 (i) performing PCR for a single-stranded sequence encoded using SD sequence to which a binding molecule is attached and ED sequence, as primers;  
 (ii) binding the binding molecule bound to the SD sequence to a substance capable of being paired up therewith, thereby recovering a PCR product;  
 (iii) denaturing the PCR product into a single strand;  
 (iv) mixing the sequences D 1 n′ labeled and D 0 n′ labeled, thereby hybridizing the single strand with the sequences D 1 n′ and D 0 n′;  
 (v) ligating the sequence D 1 n′ with the sequence D 0 n′;  
 (vi) denaturing the sequences ligated into a single-stranded sequence;  
 (vii) hybridizing sequences D 01 -D 0 n with the single-stranded sequence labeled with a marker substance, to detect or quantify the marker substance, thereby detecting or quantifying the target nucleic acid.  
 
     
     
         16 . The method according to any one of  claims 1  to  13 , wherein said first partial sequence and said second partial sequence are positioned next to each other.