Asynchronous primed PCR

Abstract

An asynchronous thermal cycling protocol for nucleic acid amplification uses two primers with thermal melting temperatures different by about 10 to 30° C. After the higher melting primer has annealed and polymerase mediated extension, the uncopied, single-stranded target sequence may be hybridized and detected by a probe. DNA probes may be cleaved by the exonuclease activity of a polymerase. The probe may be a non-cleaving analog such as PNA. When a probe is labelled with a reporter dye and a quencher selected to undergo energy transfer, e.g. FRET, fluorescence from the reporter dye may be effectively quenched when the probe is unbound. Upon hybridization of the probe to complementary target or upon cleavage while bound to target, the reporter dye is no longer quenched, resulting in a detectable amount of fluorescence. The second, lower-melting primer may be annealed and extended to generate a double-stranded nucleic acid. Amplification may be monitored in real time, including each cycle, or at the end point. The asynchronous PCR thermal cycling protocol can generate a preponderance of the PCR amplicon in single-stranded form by repetition at the end of the protocol of annealing and extension of the higher melting primer.

1 . A method of nucleic acid amplification comprising the steps of: 
 annealing a first primer to a first strand of a denatured target nucleic acid at a first annealing temperature;    extending the first primer with primer extension reagents at an extension temperature or the first annealing temperature to generate a double-stranded nucleic acid, wherein the primer extension reagents comprise a polymerase, nucleotide 5′-triphosphates, and a buffer;    annealing a detectable probe to a second strand of the denatured target nucleic acid at a probe hybridization temperature;    annealing a second primer to the second strand of the denatured target nucleic acid at a second annealing temperature wherein the second annealing temperature is lower than the first annealing temperature and extension temperature;    extending the second primer with primer extension reagents at the extension temperature to generate a double-stranded nucleic acid; and    denaturing the double-stranded target nucleic acid into a first strand and a second strand at a denaturing temperature.    
     
     
         2 . The method of  claim 1  wherein the detectable probe includes a fluorescent moiety and a quencher moiety.  
     
     
         3 . The method of  claim 2  wherein the fluorescent moiety is attached to the 5′ or 3′ terminus of the probe and the quencher moiety is attached to the 5′ or 3′ terminus of the probe.  
     
     
         4 . The method of  claim 1  wherein the probe is detected prior to extension of the second primer.  
     
     
         5 . The method of  claim 1  wherein the steps are repeated for 2 to 50 cycles.  
     
     
         6 . The method of  claim 1  wherein the probe is enzymatically cleaved.  
     
     
         7 . The method of  claim 1  wherein the probe is not enzymatically cleaved.  
     
     
         8 . The method of  claim 1  wherein the target nucleic acid is selected from a plasmid, a cDNA, an amplicon, genomic DNA, a restriction digest, and a ligation product.  
     
     
         9 . The method of  claim 1  wherein the target nucleic acid comprises single nucleotide polymorphisms.  
     
     
         10 . The method of  claim 1  wherein the first primer and second primer are DNA.  
     
     
         11 . The method of  claim 1  wherein the first primer or the second primer is a PNA/DNA chimera.  
     
     
         12 . The method of  claim 1  wherein the first primer or the second primer comprises a covalently attached fluorescent dye.  
     
     
         13 . The method of  claim 1  wherein the first primer or the second primer comprises a covalently attached mobility-modifier.  
     
     
         14 . The method of  claim 1  wherein the first primer or the second primer comprises a covalently attached minor groove binder.  
     
     
         15 . The method of  claim 1  wherein the probe comprises a target-binding sequence and two intramolecularly base-paired sequences.  
     
     
         16 . The method of  claim 15  wherein the probe forms a hairpin stem and loop structure.  
     
     
         17 . The method of  claim 15  wherein the intramolecularly base-paired sequences are at the 5′ terminus and 3′ terminus of the probe.  
     
     
         18 . The method of  claim 1  wherein the probe comprises one or more nucleotide analogs selected from a nucleobase analog, a 2′-deoxyribose analog, an internucleotide analog and an optical isomer.  
     
     
         19 . The method of  claim 18  wherein the nucleobase analog is selected from 7-deazaadenine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deaza-8-azaadenine, inosine, nebularine, nitropyrrole, nitroindole, 2-amino-purine, 2,6-diamino-purine, hypoxanthine, pseudouridine, pseudocytidine, pseudoisocytidine, 5-propynyl-cytidine, isocytidine, isoguanine, 7-deaza-quanine, 2-thio-pyrimidine, 6-thio-guanine, 4-thio-thymine, 4-thio-uracil, O 6 -methyl-guanine, N 6 -methyl-adenine, O 4 -methyl-thymine, 5,6-dihydrothymine, 5,6-dihydrouracil, 4-methyl-indole, and ethenoadenine.  
     
     
         20 . The method of  claim 18  wherein the nucleotide analog is a 2′-deoxyribose analog that is substituted at the 2′-carbon atom with Cl, F, —R, —OR, or —NR 2 , where each R is independently —H, C 1 -C 6  alkyl or C 5 -C 14  aryl.  
     
     
         21 . The method of  claim 18  wherein the nucleotide analog is an LNA.  
     
     
         22 . The method of  claim 18  wherein the nucleotide analog is an L-form optical isomer of 2′-deoxyribose.  
     
     
         23 . The method of  claim 1  wherein the probe comprises one or more 2-aminoethylglycine (PNA) monomer units.  
     
     
         24 . The method of  claim 23  wherein the probe is a PNA/DNA chimera.  
     
     
         25 . The method of  claim 1  wherein the probe has the structure:  
       
         
           
           
               
               
           
         
       
       wherein: 
 R is a fluorescent moiety;  
 L 1  and L 2  are linkers;  
 B is a nucleobase;  
 Q is a quencher moiety; and  
 n is an integer between 5 to 25.  
 
     
     
         26 . The method of  claim 25  wherein L 1  or L 2  comprise one or more amino acid units.  
     
     
         27 . The method of  claim 26  wherein L 1  and L 2  are independently selected from aspartic acid, glutamic acid, and lysine.  
     
     
         28 . The method of  claim 27  wherein L 1  is one or more aspartic acid or glutamic acid units, and L 2  is one or more lysine units.  
     
     
         29 . The method of  claim 25  wherein B is selected from uracil, thymine, cytosine, adenine, 7-deazaadenine, guanine, 7-deazaguanosine, 7-deaza-8-azaguanine, and 7-deaza-8-azaadenine.  
     
     
         30 . The method of  claim 2  wherein the fluorescent moiety is a fluorescein dye, a rhodamine dye, or a cyanine dye.  
     
     
         31 . The method of  claim 2  wherein the quencher moiety is a rhodamine dye.  
     
     
         32 . The method of  claim 2  wherein the quencher moiety is a nitro-substituted cyanine dye.  
     
     
         33 . The method of  claim 2  wherein the quencher moiety is selected from the structures:  
       
         
           
           
               
               
           
         
       
       wherein Z is selected from H, Cl, F, C 1 -C 6  alkyl, C 5 -C 14  aryl, nitro, cyano, sulfonate, NR 2 , —OR, and CO 2 H, where each R is independently H, C 1 -C 6  alkyl or C 5 -C 14  aryl.  
     
     
         34 . The method of  claim 2  wherein a change in fluorescence intensity is detected at the end-point of target amplification.  
     
     
         35 . The method of  claim 2  wherein a change in fluorescence intensity is monitored in real-time.  
     
     
         36 . The method of  claim 2  wherein a change in fluorescence intensity is detected as an indication of the presence of the target sequence.  
     
     
         37 . The method of  claim 1  wherein the first annealing temperature is 10 to 30° C. higher than the second annealing temperature.  
     
     
         38 . The method of  claim 1  wherein the first annealing temperature is 12 to 16° C. higher than the second annealing temperature.  
     
     
         39 . The method of  claim 1  wherein the first annealing temperature is 60 to 75° C.  
     
     
         40 . The method of  claim 1  wherein the second annealing temperature is 45 to 55° C.  
     
     
         41 . The method of  claim 1  wherein the first primer has a (GC) n  or a (CG) n  sequence at the 5′ terminus, where n is 1 to 4.  
     
     
         42 . The method of  claim 1  wherein a label is covalently attached to one or more of the nucleotide 5′-triphosphates at the 8-C of a purine nucleobase, the 7-C or 8-C of a 7-deazapurine nucleobase, or the 5-position of a pyrimidine nucleobase.  
     
     
         43 . The method of  claim 1  wherein a label is covalently attached to the first primer or the second primer at a 5′ terminus, a sugar, an internucleotide linkage, or a nucleobase.  
     
     
         44 . A method for producing complementary polynucleotide strands of a target polynucleotide comprising: 
 obtaining a mixture comprising first and second target polynucleotide strands which are capable of hybridizing with each other to form a base-paired structure that contains a target sequence, a first primer that is complementary to a first region in the first target polynucleotide strand, and a second primer that is complementary to a second region in the second target polynucleotide strand, such that the first and second regions flank the target sequence,    extending the first primer at a first temperature to form a first complex comprising a first complementary strand that is hybridized to the first target strand, under conditions such that the second primer does not substantially hybridize to the second region, and    extending the second primer at a second temperature that is lower than the first temperature, to form a second complex comprising a second complementary strand that is hybridized to the second target strand,    wherein before extending the second primer, a detectable probe is hybridized to a complementary binding region in the second target strand, and the hybridized probe is detected as a measure of second target strand.    
     
     
         45 . A method of nucleic acid amplification comprising the steps of: 
 annealing a first primer to a first strand of a denatured target nucleic acid at a first annealing temperature;    extending the first primer with primer extension reagents at an extension temperature or the first annealing temperature to generate a double-stranded nucleic acid, wherein the primer extension reagents comprise a polymerase, nucleotide 5′-triphosphates, and a buffer;    annealing a second primer to a second strand of the denatured target nucleic acid at a second annealing temperature wherein the second annealing temperature is lower than the first annealing temperature and extension temperature;    extending the second primer with primer extension reagents at the extension temperature to generate a double-stranded nucleic acid; and    denaturing the double-stranded target into a first strand and a second strand at a denaturing temperature.    
     
     
         46 . The method of  claim 45  wherein the steps are repeated for 2 to 50 cycles.  
     
     
         47 . The method of  claim 46  wherein the concentration of the first primer is 2 to 10 times higher than the concentration of the second primer.  
     
     
         48 . The method of  claim 46  wherein the steps of annealing the second primer to the second strand of the denatured target and extending the second primer are omitted in the last 1-25 cycles, whereby a mixture of single-stranded and double-stranded DNA is produced.  
     
     
         49 . The method of  claim 46  wherein the steps of annealing the second primer to the second strand of the denatured target and extending the second primer are omitted in the last 1-10 cycles so as to produce a preponderance of ss DNA.  
     
     
         50 . The method of  claim 45  wherein the target is a cDNA.  
     
     
         51 . The method of  claim 45  wherein the first primer is labelled with a fluorescent dye.  
     
     
         52 . The method of  claim 45  further comprising the step of hybridizing the single-stranded and double-stranded DNA product mixture to a plurality of probes immobilized on an array.  
     
     
         53 . The method of  claim 52  wherein the probes are FRET probes.  
     
     
         54 . A method for producing complementary polynucleotide strands of a target polynucleotide comprising: 
 obtaining a mixture comprising a first and second target polynucleotides which are capable of hybridizing with each other to form a base-paired structure that contains a target sequence, a first primer that is complementary to a first region in the first target polynucleotide, and a second primer that is complementary to a second region in the second target polynucleotide, such that the first and second regions flank the target sequence,    extending the first primer at a first temperature to form a first complex comprising a first complementary strand that is hybridized to the first target strand, under conditions such that the second primer does not substantially hybridize to the second region, and    extending the second primer at a second temperature that is lower than the first temperature, to form a second complex comprising a second complementary strand that is hybridized to the second target strand.    
     
     
         55 . The method of  claim 54 , which further comprises denaturing the first and second complexes after the second primer has been extended.  
     
     
         56 . The method of  claim 55 , which further comprises repeating the first primer extension, second primer extension, and denaturation steps in one or more cycles.  
     
     
         57 . The method of  claim 55 , wherein after said denaturation, first primer is extended at the first temperature to form a mixture comprising the second target polynucleotide in single-stranded form and the first complex in duplex form.  
     
     
         58 . A kit for amplifying a target polynucleotide comprising two or more primers, wherein a first primer and a second primer have a Tm difference of 10 to 30° C.  
     
     
         59 . The kit of  claim 58  wherein a said primer is labelled with a fluorescent dye.  
     
     
         60 . The kit of  claim 58  further comprising a polymerase.  
     
     
         61 . The kit of  claim 58  further comprising a detectable probe.  
     
     
         62 . The kit of  claim 61  wherein the detectable probe is DNA and the probe includes a fluorescent moiety and a quencher moiety.  
     
     
         63 . The kit of  claim 61  wherein the detectable probe is PNA and the probe includes a fluorescent moiety and a quencher moiety.  
     
     
         64 . The kit of  claim 61  wherein the probe comprises a nucleic acid analog selected from a nucleobase analog, a 2′-deoxyribose analog, an internucleotide analog and an optical isomer.  
     
     
         65 . The kit of  claim 58  further comprising one or more enzymatically-extendable nucleotides.  
     
     
         66 . The kit of  claim 65  wherein a nucleotide is labelled with a fluorescent dye.