CA2425551A1 - Procede, construct et conjugue permettant de produire des copies multip les d'acides nucleiques - Google Patents
Procede, construct et conjugue permettant de produire des copies multip les d'acides nucleiques Download PDFInfo
- Publication number
- CA2425551A1 CA2425551A1 CA002425551A CA2425551A CA2425551A1 CA 2425551 A1 CA2425551 A1 CA 2425551A1 CA 002425551 A CA002425551 A CA 002425551A CA 2425551 A CA2425551 A CA 2425551A CA 2425551 A1 CA2425551 A1 CA 2425551A1
- Authority
- CA
- Canada
- Prior art keywords
- nucleic acid
- construct
- conjugate
- protein
- promoter
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 208
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 200
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 199
- 238000000034 method Methods 0.000 title claims abstract description 85
- 230000008569 process Effects 0.000 title claims abstract description 48
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 42
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 claims abstract description 36
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 claims abstract description 35
- 230000000295 complement effect Effects 0.000 claims abstract description 34
- 238000013518 transcription Methods 0.000 claims abstract description 19
- 230000035897 transcription Effects 0.000 claims abstract description 19
- 238000001727 in vivo Methods 0.000 claims abstract description 17
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 14
- 238000013519 translation Methods 0.000 claims abstract description 6
- 102000044158 nucleic acid binding protein Human genes 0.000 claims abstract description 4
- 108700020942 nucleic acid binding protein Proteins 0.000 claims abstract description 4
- 102000005962 receptors Human genes 0.000 claims abstract description 3
- 239000003446 ligand Substances 0.000 claims abstract 2
- 108020003175 receptors Proteins 0.000 claims abstract 2
- 108020004414 DNA Proteins 0.000 claims description 101
- 125000003729 nucleotide group Chemical group 0.000 claims description 48
- 102000004190 Enzymes Human genes 0.000 claims description 33
- 108090000790 Enzymes Proteins 0.000 claims description 33
- 102000004169 proteins and genes Human genes 0.000 claims description 28
- 102000053602 DNA Human genes 0.000 claims description 21
- 230000000977 initiatory effect Effects 0.000 claims description 21
- 238000006116 polymerization reaction Methods 0.000 claims description 16
- 102100034343 Integrase Human genes 0.000 claims description 13
- 229920002477 rna polymer Polymers 0.000 claims description 10
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 9
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 9
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 7
- 230000000692 anti-sense effect Effects 0.000 claims description 7
- 229920001577 copolymer Polymers 0.000 claims description 6
- 229920000642 polymer Polymers 0.000 claims description 5
- 230000000536 complexating effect Effects 0.000 claims description 4
- 238000011144 upstream manufacturing Methods 0.000 claims description 3
- 102000009661 Repressor Proteins Human genes 0.000 claims 1
- 108010034634 Repressor Proteins Proteins 0.000 claims 1
- 238000000338 in vitro Methods 0.000 abstract description 28
- 239000000872 buffer Substances 0.000 abstract description 21
- 230000037452 priming Effects 0.000 abstract description 10
- 239000013615 primer Substances 0.000 description 134
- 230000003321 amplification Effects 0.000 description 56
- 238000003199 nucleic acid amplification method Methods 0.000 description 56
- 238000006243 chemical reaction Methods 0.000 description 47
- 238000004519 manufacturing process Methods 0.000 description 43
- 239000000047 product Substances 0.000 description 40
- 239000002773 nucleotide Substances 0.000 description 31
- 239000002243 precursor Substances 0.000 description 29
- QTANTQQOYSUMLC-UHFFFAOYSA-O Ethidium cation Chemical compound C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 QTANTQQOYSUMLC-UHFFFAOYSA-O 0.000 description 26
- 239000000203 mixture Substances 0.000 description 25
- 239000000523 sample Substances 0.000 description 23
- 108091034117 Oligonucleotide Proteins 0.000 description 22
- 238000009396 hybridization Methods 0.000 description 22
- 210000004027 cell Anatomy 0.000 description 21
- 230000000694 effects Effects 0.000 description 20
- -1 nucleoside triphosphates Chemical class 0.000 description 20
- 230000015572 biosynthetic process Effects 0.000 description 19
- 238000010348 incorporation Methods 0.000 description 18
- 239000011541 reaction mixture Substances 0.000 description 16
- 239000002777 nucleoside Substances 0.000 description 15
- 238000003786 synthesis reaction Methods 0.000 description 15
- 239000012634 fragment Substances 0.000 description 14
- 238000002474 experimental method Methods 0.000 description 13
- 239000013612 plasmid Substances 0.000 description 13
- 239000001226 triphosphate Substances 0.000 description 13
- 235000011178 triphosphate Nutrition 0.000 description 13
- 239000003054 catalyst Substances 0.000 description 12
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 10
- 244000309466 calf Species 0.000 description 10
- 239000011543 agarose gel Substances 0.000 description 9
- 230000004048 modification Effects 0.000 description 9
- 238000012986 modification Methods 0.000 description 9
- 230000010076 replication Effects 0.000 description 9
- 210000001541 thymus gland Anatomy 0.000 description 9
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 8
- 102000040430 polynucleotide Human genes 0.000 description 8
- 108091033319 polynucleotide Proteins 0.000 description 8
- 108091093088 Amplicon Proteins 0.000 description 7
- 101710137500 T7 RNA polymerase Proteins 0.000 description 7
- 230000001419 dependent effect Effects 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 238000002844 melting Methods 0.000 description 7
- 230000008018 melting Effects 0.000 description 7
- 230000000379 polymerizing effect Effects 0.000 description 7
- 239000002157 polynucleotide Substances 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 101710203526 Integrase Proteins 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 6
- 238000006073 displacement reaction Methods 0.000 description 6
- 238000005755 formation reaction Methods 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 239000000499 gel Substances 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 108091008146 restriction endonucleases Proteins 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 102000004594 DNA Polymerase I Human genes 0.000 description 5
- 229960002685 biotin Drugs 0.000 description 5
- 235000020958 biotin Nutrition 0.000 description 5
- 239000011616 biotin Substances 0.000 description 5
- 239000012954 diazonium Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-O diazynium Chemical compound [NH+]#N IJGRMHOSHXDMSA-UHFFFAOYSA-O 0.000 description 5
- 230000029087 digestion Effects 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- VVJKKWFAADXIJK-UHFFFAOYSA-N Allylamine Chemical compound NCC=C VVJKKWFAADXIJK-UHFFFAOYSA-N 0.000 description 4
- 108090001008 Avidin Proteins 0.000 description 4
- 108010017826 DNA Polymerase I Proteins 0.000 description 4
- 229910019142 PO4 Inorganic materials 0.000 description 4
- 238000002105 Southern blotting Methods 0.000 description 4
- 238000001668 nucleic acid synthesis Methods 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical group [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 4
- 239000010452 phosphate Substances 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 108020005544 Antisense RNA Proteins 0.000 description 3
- 108020004635 Complementary DNA Proteins 0.000 description 3
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 3
- XKMLYUALXHKNFT-UUOKFMHZSA-N Guanosine-5'-triphosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XKMLYUALXHKNFT-UUOKFMHZSA-N 0.000 description 3
- 108020004682 Single-Stranded DNA Proteins 0.000 description 3
- 108010006785 Taq Polymerase Proteins 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 239000003184 complementary RNA Substances 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 239000003365 glass fiber Substances 0.000 description 3
- 238000009830 intercalation Methods 0.000 description 3
- 238000007834 ligase chain reaction Methods 0.000 description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 230000002285 radioactive effect Effects 0.000 description 3
- 230000001360 synchronised effect Effects 0.000 description 3
- 230000010474 transient expression Effects 0.000 description 3
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- UFBJCMHMOXMLKC-UHFFFAOYSA-N 2,4-dinitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O UFBJCMHMOXMLKC-UHFFFAOYSA-N 0.000 description 2
- 108091027075 5S-rRNA precursor Proteins 0.000 description 2
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 description 2
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 2
- 108020004491 Antisense DNA Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 239000003155 DNA primer Substances 0.000 description 2
- 230000006820 DNA synthesis Effects 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 102100031780 Endonuclease Human genes 0.000 description 2
- 108010042407 Endonucleases Proteins 0.000 description 2
- 101000686777 Escherichia phage T7 T7 RNA polymerase Proteins 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 102000003960 Ligases Human genes 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 2
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 2
- 102000001218 Rec A Recombinases Human genes 0.000 description 2
- 108010055016 Rec A Recombinases Proteins 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- RZCIEJXAILMSQK-JXOAFFINSA-N TTP Chemical compound O=C1NC(=O)C(C)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 RZCIEJXAILMSQK-JXOAFFINSA-N 0.000 description 2
- 239000007984 Tris EDTA buffer Substances 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 229960001456 adenosine triphosphate Drugs 0.000 description 2
- 238000005054 agglomeration Methods 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 239000003816 antisense DNA Substances 0.000 description 2
- 102000023732 binding proteins Human genes 0.000 description 2
- 108091008324 binding proteins Proteins 0.000 description 2
- 230000008827 biological function Effects 0.000 description 2
- 108700021042 biotin binding protein Proteins 0.000 description 2
- 102000043871 biotin binding protein Human genes 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000007385 chemical modification Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 230000006862 enzymatic digestion Effects 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 238000002189 fluorescence spectrum Methods 0.000 description 2
- 125000005842 heteroatom Chemical group 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000000138 intercalating agent Substances 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 108010026228 mRNA guanylyltransferase Proteins 0.000 description 2
- 239000011777 magnesium Substances 0.000 description 2
- 229910052749 magnesium Inorganic materials 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000002853 nucleic acid probe Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 230000029058 respiratory gaseous exchange Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 229910052717 sulfur Inorganic materials 0.000 description 2
- 150000003573 thiols Chemical class 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 125000002264 triphosphate group Chemical class [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 235000011330 Armoracia rusticana Nutrition 0.000 description 1
- 240000003291 Armoracia rusticana Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- PCDQPRRSZKQHHS-UHFFFAOYSA-N Cytidine 5'-triphosphate Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 PCDQPRRSZKQHHS-UHFFFAOYSA-N 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- HAEJPQIATWHALX-KQYNXXCUSA-N ITP Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O[C@H]1N1C(N=CNC2=O)=C2N=C1 HAEJPQIATWHALX-KQYNXXCUSA-N 0.000 description 1
- 239000007993 MOPS buffer Substances 0.000 description 1
- 108060004795 Methyltransferase Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
- 108010066717 Q beta Replicase Proteins 0.000 description 1
- 108020004518 RNA Probes Proteins 0.000 description 1
- 239000013616 RNA primer Substances 0.000 description 1
- 239000003391 RNA probe Substances 0.000 description 1
- 230000006819 RNA synthesis Effects 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 241000589596 Thermus Species 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- PGAVKCOVUIYSFO-XVFCMESISA-N UTP Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 PGAVKCOVUIYSFO-XVFCMESISA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001745 anti-biotin effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 108010028263 bacteriophage T3 RNA polymerase Proteins 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- PCDQPRRSZKQHHS-ZAKLUEHWSA-N cytidine-5'-triphosphate Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO[P@](O)(=O)O[P@@](O)(=O)OP(O)(O)=O)O1 PCDQPRRSZKQHHS-ZAKLUEHWSA-N 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-N dCTP Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](CO[P@](O)(=O)O[P@](O)(=O)OP(O)(O)=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-N 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 230000001687 destabilization Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000013024 dilution buffer Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 230000026030 halogenation Effects 0.000 description 1
- 238000005658 halogenation reaction Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000007169 ligase reaction Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 229940046166 oligodeoxynucleotide Drugs 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 229910000028 potassium bicarbonate Inorganic materials 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 150000003141 primary amines Chemical class 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 210000004915 pus Anatomy 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- XKMLYUALXHKNFT-UHFFFAOYSA-N rGTP Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O XKMLYUALXHKNFT-UHFFFAOYSA-N 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 210000003802 sputum Anatomy 0.000 description 1
- 208000024794 sputum Diseases 0.000 description 1
- 125000000446 sulfanediyl group Chemical group *S* 0.000 description 1
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 210000001179 synovial fluid Anatomy 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000005382 thermal cycling Methods 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 239000005451 thionucleotide Substances 0.000 description 1
- 238000010490 three component reaction Methods 0.000 description 1
- 238000006257 total synthesis reaction Methods 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- PGAVKCOVUIYSFO-UHFFFAOYSA-N uridine-triphosphate Natural products OC1C(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)OC1N1C(=O)NC(=O)C=C1 PGAVKCOVUIYSFO-UHFFFAOYSA-N 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 101150069452 z gene Proteins 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US18262194A | 1994-01-13 | 1994-01-13 | |
| US08/182,621 | 1994-01-13 | ||
| CA002140081A CA2140081C (fr) | 1994-01-13 | 1995-01-12 | Procede, construct et conjugue permettant de produire des copies multiples d'acides nucleiques |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002140081A Division CA2140081C (fr) | 1994-01-13 | 1995-01-12 | Procede, construct et conjugue permettant de produire des copies multiples d'acides nucleiques |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2425551A1 true CA2425551A1 (fr) | 1995-07-14 |
Family
ID=25677731
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002425551A Abandoned CA2425551A1 (fr) | 1994-01-13 | 1995-01-12 | Procede, construct et conjugue permettant de produire des copies multip les d'acides nucleiques |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2425551A1 (fr) |
-
1995
- 1995-01-12 CA CA002425551A patent/CA2425551A1/fr not_active Abandoned
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2140081C (fr) | Procede, construct et conjugue permettant de produire des copies multiples d'acides nucleiques | |
| US20010000077A1 (en) | Novel process, construct and conjugate for producing multiple nucleic acid copies | |
| KR960015744B1 (ko) | 핵산을 증폭시키는 방법 | |
| JP2648802B2 (ja) | 増強された核酸増幅方法 | |
| JP2807202B2 (ja) | 核酸の高感度検出方法 | |
| US5322770A (en) | Reverse transcription with thermostable DNA polymerases - high temperature reverse transcription | |
| US6180338B1 (en) | Method, reagent and kit for the detection and amplification of nucleic acid sequences | |
| JP3330946B2 (ja) | 一本鎖ｄｎａ分子の生成方法 | |
| JP3515108B2 (ja) | 二本鎖ｒｎａの製法とその応用 | |
| JP3514630B2 (ja) | 核酸配列の増幅および検出 | |
| AU2002329104B2 (en) | Annealing control primer and its uses | |
| DK2365078T3 (en) | Methods using the dual specificity oligonucleotide and dual specificity oligonucleotide | |
| JP3403405B2 (ja) | 核酸伸長検定 | |
| JPH10510161A (ja) | 末端反復増幅方法 | |
| KR19980018395A (ko) | 변형된 열안정성 디엔에이 폴리머라아제 | |
| CA2467577A1 (fr) | Pcr multiplex | |
| JP2002291490A (ja) | 修飾プライマーを使用する増幅 | |
| AU2329497A (en) | Target nucleic acid sequence amplification | |
| JPH0759599A (ja) | 標的ｒｎａ分子の増幅方法 | |
| AU782555B2 (en) | Compositions and methods for enhanced sensitivity and specificity of nucleic acid synthesis | |
| CN110603326A (zh) | 扩增靶核酸的方法 | |
| US6986985B1 (en) | Process for producing multiple nucleic acid copies in vivo using a protein-nucleic acid construct | |
| JP5436105B2 (ja) | 改善された核酸増幅のためのポリアニオン | |
| WO1990001064A1 (fr) | Techniques d'amplification selectve de sequences | |
| EP0598832A1 (fr) | Sequencage de l'adn avec une exonuclease d'un gene 6 de type t7 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| EEER | Examination request |
Effective date: 20030424 |
|
| FZDE | Dead |
Effective date: 20130403 |