Selective Amplfication of Nucleic Acid Sequences

Abstract

The present invention relates to the field of nucleic acid sequence replication including PCR. Specifically, the present invention relates to methods and compositions for amplifying one or more target sequences from one or more template sequences. In particular, the present invention provides novel primer designs to enhance specificity of PCR reactions. The present invention also provides methods and compositions to perform the selection of specific sequence sections using specific primers and the amplification of all selected sequence sections using a pair of common primers in a single reaction tube.

What is claimed is: 
     
         1 . An oligonucleotide primer comprising a 3p arm having a 3′ end and a 5′ end, a loop section and a 5p arm having a 3′ end and a 5′ end, wherein the 5p arm hybridizes to a DNA template and wherein the 3p arm hybridizes to the DNA template and provides sequence specificity for polymerase extension and wherein the loop section is located between the 5p arm and the 3p arm and does not bind the DNA template. 
     
     
         2 . The primer of  claim 1  wherein the DNA template is substantially complementary to the 5p arm and the 3p arm. 
     
     
         3 . The primer of  claim 1  wherein the 5p arm is from 10 to 100 nucleotides in length. 
     
     
         4 . The primer of  claim 1  wherein the 3p arm is from 6 to 60 nucleotides in length. 
     
     
         5 . The primer of  claim 1  wherein the loop section is from 12 to 50 nucleotides in length. 
     
     
         6 . The primer of  claim 1  wherein the 5p is from 25 to 60 nucleotides in length and wherein the 3p arm is from 10 to 20 nucleotides in length and wherein the loop section is from 15 to 40 nucleotides in length. 
     
     
         7 . The primer of  claim 1  wherein the 5′ end of the 3p arm and the 3′ end of the 5p arm are adjacent each other when bound to the DNA template. 
     
     
         8 . The primer of  claim 1  wherein the 5p arm has higher binding energy than the 3p arm when hybridized to the DNA template. 
     
     
         9 . The primer of  claim 1  wherein the 5p arm has a binding energy higher than twice that of the 3p arm. 
     
     
         10 . The primer of  claim 1  wherein the 5p arm has a binding energy higher than three times that of the 3p arm. 
     
     
         11 . The primer of  claim 1  wherein the loop section is a bulge loop. 
     
     
         12 . The primer of  claim 1  wherein the loop section is a hairpin loop. 
     
     
         13 . The primer of  claim 1  wherein the loop section is an internal loop. 
     
     
         14 . A hybridization structure for assay use comprising a probe and a target wherein the hybridization structure has one or more single stranded loops and two or more duplex segments wherein each loop is located between the duplex segments. 
     
     
         15 . The hybridization structure of  claim 14  wherein the single stranded loop is in the probe and comprises one or more non-nucleotide moieties. 
     
     
         16 . The hybridization structure of  claim 14  wherein the probe comprises a spacer. 
     
     
         17 . The hybridization structure of  claim 14  wherein the hybridization structure is used for polymerase extension. 
     
     
         18 . The hybridization structure of  claim 14  wherein the hybridization structure is used for hybridization detection. 
     
     
         19 . The hybridization structure of  claim 18  wherein the loop contains 12 to 50 nucleotides. 
     
     
         20 . A method of amplifying a target nucleic acid comprising:
 providing a first specific primer, a first common primer, a second common primer, a flanked target fragment, a polymerase and nucleotides;   performing a target selection comprising one cycle of a first thermocycling routine comprising an denaturation step, annealing step and an extension step; and,   performing amplification comprising two or more cycles of a second thermocycling routine comprising an denaturation step, annealing step and an extension step thereby amplifying the target nucleic acid.   
     
     
         21 . A method of amplifying a target nucleic acid comprising:
 providing a first specific primer, a second specific primer, a first common primer, a second common primer, a target nucleic acid, a polymerase and nucleotides;   performing a target selection comprising two cycles of a first thermocycling routine comprising an denaturation step, annealing step and an extension step; and,   performing amplification comprising two or more cycles of a second thermocycling routine comprising an denaturation step, annealing step and an extension step thereby amplifying the target nucleic acid.   
     
     
         22 . The method of  claim 20  or  21  wherein the first specific primer has a 3′ end and a 5′ end wherein the 3′ end contains a first sequence specific segment and the 5′ end contains a first common segment. 
     
     
         23 . The method of  claim 20  or  21  wherein the second specific primer has a 3′ end and a 5′ end wherein the 3′ end contains a second sequence specific segment and the 5′ end contains a second common segment. 
     
     
         26 . The method of  claim 20  or  21  wherein the concentration of the first and second specific primers is 500 fold less than that of the first and second common primer. 
     
     
         27 . The method of  claim 20  or  21  wherein the concentration of the first and second specific primers is from about 0.0001 nM to about 5 nM. 
     
     
         28 . The method of  claim 20  or  21  wherein the concentration of the first and second common primers is from about 200 nM to about 5000 nM. 
     
     
         29 . The method of  claim 20  or  21  wherein the concentration of the first and second specific primers is less than 1 nM. 
     
     
         30 . The method of  claim 20  or  21  wherein the concentration of the first and second common primers is more than 200 nM. 
     
     
         31 . The method of  claim 20  or  21  wherein the first specific primer is an omega primer and wherein the second specific primer is an omega primer. 
     
     
         32 . The method of  claim 20  or  21  wherein the annealing time for the first thermocycling routine is from about 30 minute to about 4 hours. 
     
     
         33 . The method of  claim 20  or  21  wherein the annealing temperature for the first thermocycling routine is from about 60° C. to about 75° C. 
     
     
         34 . The method of  claim 20  or  21  wherein the annealing temperature for the first thermocycling routine is from about 60° C. to about 72° C. 
     
     
         35 . The method of  claim 20  or  21  wherein the annealing temperature for the first thermocycling routine is from about 65° C. to about 72° C. 
     
     
         36 . The method of  claim 20  or  21  wherein the second thermocycling routine has from 10-50 cycles. 
     
     
         37 . The method of  claim 20  or  21  wherein the second thermocycling routine has from 20-40 cycles. 
     
     
         38 . The method of  claim 20  or  21 , wherein the annealing temperature is within 10° C. of the peak polymerase activity of the polymerase. 
     
     
         39 . The method of  claim 20  or  21 , wherein the polymerase is a polymerase without strand-displacement activity and 5′ to 3′ nuclease activity. 
     
     
         40 . The method of  claim 20  or  21 , wherein the polymerase is selected from the group consisting of Phusion Hot Start Flex DNA polymerase and Q5® Hot Start High-Fidelity DNA Polymerase. 
     
     
         41 . A method of amplifying two or more different target nucleic acids comprising:
 providing a set of specific primer pairs wherein each pair comprises a first specific primer and a second specific primer and is designed for a specific target nucleic acid, a first common primer, a second common primer and a set of target nucleic acids, a polymerase and nucleotides;   performing two cycles of a first thermocycling routine comprising an denaturation step, annealing step and an extension step; and   performing two or more cycles of a second thermocycling routine comprising an denaturation step, annealing step and an extension step thereby amplifying the target nucleic acid.   
     
     
         42 . The method of  claim 41  wherein the concentration of the first and second specific primers is 500 fold less than that of the first and second common primer. 
     
     
         43 . The method of  claim 41  wherein the concentration of the first and second specific primers is from about 0.0001 nM to about 5 nM. 
     
     
         44 . The method of  claim 41  wherein the concentration of the first and second common primers is from about 200 nM to about 5000 nM. 
     
     
         45 . The method of  claim 41  wherein the concentration of the first and second specific primers is less than 1 nM. 
     
     
         46 . The method of  claim 41  wherein the concentration of the first and second common primers is more than 500 nM. 
     
     
         47 . The method of  claim 41  wherein the annealing time for the first thermocycling routine is from about 30 minute to about 4 hours. 
     
     
         48 . The method of  claim 41  wherein the first specific primer is an omega primer and wherein the second specific primer is an omega primer. 
     
     
         49 . The method of  claim 41  wherein the first specific primer is a regular specific primer and wherein the second specific primer is a regular specific primer. 
     
     
         50 . A method of purifying PCR products comprising adding, to a mixture of PCR reaction components comprising target sequences, a first common primer and a second common primer, DNA fragments, polymerase, PCR buffer solution wherein the target sequences are flanked with priming segments that are either identical or complementary to the first common primer and the second common primer, the fragments do not contain priming segments, and the second common primer comprises a priming segment, a modifier segment and a tag segment; probe grafted beads wherein the probe has a sequence that is substantially complementary to that of tag segment and facilitates the capture of the PCR product by the beads through hybridization. 
     
     
         51 . The method of  claim 50  Wherein the modifier segment is selected from the group consisting of one or more C3 alkyl spacers, one or more ethylene glycol spacers, one or more photo-cleavable spacers, one or more 1′,2′-dideoxyribose, one or more deoxyuridines or combinations thereof 
     
     
         52 . The method of  claim 50 , wherein the tag segment comprises of at least one binding moiety. 
     
     
         53 . The method of  claim 52 , wherein the binding moiety is biotin. 
     
     
         54 . The method of  claim 50 , wherein the tag segment comprises an oligonucleotide and a binding moiety. 
     
     
         55 . The method of  claim 52 , wherein the binding moiety is attached to the 5′ end of the tag segment oligonucleotide. 
     
     
         56 . A method of generating surface clusters comprising
 amplifying a target sequence with a first common primer and a second common primer wherein the first common primer comprising a priming segment and the second common primer comprising a priming segment, modifier segment and tag segment to produce a PCR product containing a single-stranded tag wherein the PCR product comprises the first strand and the second strand wherein the second strand is connected to the single-stranded tag;   providing a substrate wherein the substrate comprises a probe, a first surface primer and a second surface primer;   applying the PCR product and a guide to the substrate thereby hybridizing the PCR product, the guide and the probe to produce a PCR product/guide/probe complex on the substrate surface;   ligating the PCR product and the probe thereby linking the PCR product;   washing the substrate thereby removing the first strand of the PCR product; and,   extending the first surface primer and the second surface primer thereby forming surface clusters.   
     
     
         57 . The method of  claim 56  wherein a probe, a first surface primer, and a second surface primer are attached to the substrate. 
     
     
         58 . The method of  claim 57  wherein the probe, a first surface primer, and a second surface prime further comprise a spacer through which the probe, the first surface primer, and the second surface prime are connected to the substrate surface. 
     
     
         59 . The method of any one of  claims 20 ,  21 ,  41 ,  50  or  56  wherein the common primer has a tail segment and a common segment. 
     
     
         60 . The method of any one of  claims 20 ,  21  or  41  wherein the specific primer has a specific segment and a common segment. 
     
     
         60 . A method of designing a PCR primer, the method comprising:
 a) determining a primer length to produce a sufficient template association coefficient;   b) determining primer 3′ end binding coefficient;   c) determining template association coefficient in the presence of folding effect; and   d) determining priming efficiency by combining association coefficient of variant alleles.   
     
     
         61 . The primer of any one of  claims 1 - 13  having a common segment and a specific segment, wherein the specific segment is comprised of the 3p arm and 5p arm and wherein the loop is comprised of the common segment