CA2453864A1 - A walk-through technique for in vitro recombination of polynucleotide sequences - Google Patents
A walk-through technique for in vitro recombination of polynucleotide sequences Download PDFInfo
- Publication number
- CA2453864A1 CA2453864A1 CA002453864A CA2453864A CA2453864A1 CA 2453864 A1 CA2453864 A1 CA 2453864A1 CA 002453864 A CA002453864 A CA 002453864A CA 2453864 A CA2453864 A CA 2453864A CA 2453864 A1 CA2453864 A1 CA 2453864A1
- Authority
- CA
- Canada
- Prior art keywords
- dna
- nucleic acid
- chain
- molecules
- template
- 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
- 238000000034 method Methods 0.000 title claims abstract description 63
- 102000040430 polynucleotide Human genes 0.000 title claims abstract description 38
- 108091033319 polynucleotide Proteins 0.000 title claims abstract description 38
- 239000002157 polynucleotide Substances 0.000 title claims abstract description 38
- 238000005215 recombination Methods 0.000 title abstract description 28
- 230000006798 recombination Effects 0.000 title abstract description 28
- 238000000338 in vitro Methods 0.000 title abstract description 11
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 46
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 19
- 230000003321 amplification Effects 0.000 claims abstract description 10
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 10
- 239000012634 fragment Substances 0.000 claims description 74
- 102000004190 Enzymes Human genes 0.000 claims description 28
- 108090000790 Enzymes Proteins 0.000 claims description 28
- 150000007523 nucleic acids Chemical group 0.000 claims description 28
- 239000000203 mixture Substances 0.000 claims description 23
- 230000000694 effects Effects 0.000 claims description 20
- 102000039446 nucleic acids Human genes 0.000 claims description 17
- 108020004707 nucleic acids Proteins 0.000 claims description 17
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 14
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 14
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 10
- 239000011541 reaction mixture Substances 0.000 claims description 10
- 239000013598 vector Substances 0.000 claims description 10
- 238000010367 cloning Methods 0.000 claims description 9
- 238000012216 screening Methods 0.000 claims description 8
- 238000001668 nucleic acid synthesis Methods 0.000 claims description 5
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 4
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 4
- 229920001184 polypeptide Polymers 0.000 claims 3
- 238000010187 selection method Methods 0.000 claims 1
- 244000309466 calf Species 0.000 abstract description 31
- 101710184243 Intestinal-type alkaline phosphatase Proteins 0.000 abstract description 28
- 102100024319 Intestinal-type alkaline phosphatase Human genes 0.000 abstract description 26
- 230000003169 placental effect Effects 0.000 abstract description 24
- 108010060059 Sarcosine Oxidase Proteins 0.000 abstract description 5
- 102000008118 Sarcosine oxidase Human genes 0.000 abstract description 3
- 238000002703 mutagenesis Methods 0.000 abstract description 3
- 231100000350 mutagenesis Toxicity 0.000 abstract description 3
- 229910019142 PO4 Inorganic materials 0.000 abstract 1
- 235000021317 phosphate Nutrition 0.000 abstract 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 122
- 238000006243 chemical reaction Methods 0.000 description 36
- 238000003786 synthesis reaction Methods 0.000 description 32
- 230000015572 biosynthetic process Effects 0.000 description 30
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 26
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 25
- 239000000872 buffer Substances 0.000 description 20
- 230000035772 mutation Effects 0.000 description 17
- 239000000047 product Substances 0.000 description 15
- 238000012163 sequencing technique Methods 0.000 description 15
- 102000053602 DNA Human genes 0.000 description 11
- 239000002773 nucleotide Substances 0.000 description 11
- 125000003729 nucleotide group Chemical group 0.000 description 11
- 238000005457 optimization Methods 0.000 description 11
- 108091028043 Nucleic acid sequence Proteins 0.000 description 9
- 230000001351 cycling effect Effects 0.000 description 9
- 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 9
- 230000006870 function Effects 0.000 description 9
- 108060002716 Exonuclease Proteins 0.000 description 8
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 8
- 238000013459 approach Methods 0.000 description 8
- 239000005546 dideoxynucleotide Substances 0.000 description 8
- 102000013165 exonuclease Human genes 0.000 description 8
- 108010031345 placental alkaline phosphatase Proteins 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 102100024321 Alkaline phosphatase, placental type Human genes 0.000 description 6
- 230000006820 DNA synthesis Effects 0.000 description 6
- 102100031780 Endonuclease Human genes 0.000 description 6
- 238000000137 annealing Methods 0.000 description 6
- 230000000295 complement effect Effects 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 108010017826 DNA Polymerase I Proteins 0.000 description 5
- 102000004594 DNA Polymerase I Human genes 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 5
- 108010072685 Uracil-DNA Glycosidase Proteins 0.000 description 5
- 102000006943 Uracil-DNA Glycosidase Human genes 0.000 description 5
- 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 5
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 5
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 5
- 108010052305 exodeoxyribonuclease III Proteins 0.000 description 5
- 238000013467 fragmentation Methods 0.000 description 5
- 238000006062 fragmentation reaction Methods 0.000 description 5
- 101150028668 APO1 gene Proteins 0.000 description 4
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 4
- 235000014469 Bacillus subtilis Nutrition 0.000 description 4
- 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 4
- 101000962448 Malus domestica Major allergen Mal d 1 Proteins 0.000 description 4
- 108010006785 Taq Polymerase Proteins 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 238000003776 cleavage reaction Methods 0.000 description 4
- 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 4
- 229910001629 magnesium chloride Inorganic materials 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- FSYKKLYZXJSNPZ-UHFFFAOYSA-N sarcosine Chemical compound C[NH2+]CC([O-])=O FSYKKLYZXJSNPZ-UHFFFAOYSA-N 0.000 description 4
- 230000007017 scission Effects 0.000 description 4
- 238000002864 sequence alignment Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- OAKPWEUQDVLTCN-NKWVEPMBSA-N 2',3'-Dideoxyadenosine-5-triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1CC[C@@H](CO[P@@](O)(=O)O[P@](O)(=O)OP(O)(O)=O)O1 OAKPWEUQDVLTCN-NKWVEPMBSA-N 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- 241000713838 Avian myeloblastosis virus Species 0.000 description 3
- 244000063299 Bacillus subtilis Species 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 241000701533 Escherichia virus T4 Species 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 241000208445 Sarcodes Species 0.000 description 3
- HDRRAMINWIWTNU-NTSWFWBYSA-N [[(2s,5r)-5-(2-amino-6-oxo-3h-purin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@H]1CC[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HDRRAMINWIWTNU-NTSWFWBYSA-N 0.000 description 3
- ARLKCWCREKRROD-POYBYMJQSA-N [[(2s,5r)-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)CC1 ARLKCWCREKRROD-POYBYMJQSA-N 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 210000004899 c-terminal region Anatomy 0.000 description 3
- 238000012219 cassette mutagenesis Methods 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- URGJWIFLBWJRMF-JGVFFNPUSA-N ddTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)CC1 URGJWIFLBWJRMF-JGVFFNPUSA-N 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000001502 gel electrophoresis Methods 0.000 description 3
- 238000010348 incorporation Methods 0.000 description 3
- 230000000968 intestinal effect Effects 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 238000002708 random mutagenesis Methods 0.000 description 3
- 239000011535 reaction buffer Substances 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 239000001226 triphosphate Substances 0.000 description 3
- 235000011178 triphosphate Nutrition 0.000 description 3
- 125000002264 triphosphate group Chemical group [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 0.000 description 3
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 3
- 208000035657 Abasia Diseases 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 238000001712 DNA sequencing Methods 0.000 description 2
- 241000701832 Enterobacteria phage T3 Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108091005461 Nucleic proteins Proteins 0.000 description 2
- 108010002747 Pfu DNA polymerase Proteins 0.000 description 2
- 108010010677 Phosphodiesterase I Proteins 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 108010077895 Sarcosine Proteins 0.000 description 2
- 108020004682 Single-Stranded DNA Proteins 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 108010058966 bacteriophage T7 induced DNA polymerase Proteins 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 238000004422 calculation algorithm Methods 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 229940023064 escherichia coli Drugs 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- KWIUHFFTVRNATP-UHFFFAOYSA-N glycine betaine Chemical compound C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229940046166 oligodeoxynucleotide Drugs 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 230000004853 protein function Effects 0.000 description 2
- 230000002797 proteolythic effect Effects 0.000 description 2
- 229940043230 sarcosine Drugs 0.000 description 2
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- CZVCGJBESNRLEQ-UHFFFAOYSA-N 7h-purine;pyrimidine Chemical class C1=CN=CN=C1.C1=NC=C2NC=NC2=N1 CZVCGJBESNRLEQ-UHFFFAOYSA-N 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 108020001738 DNA Glycosylase Proteins 0.000 description 1
- 102000028381 DNA glycosylase Human genes 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 102000052510 DNA-Binding Proteins Human genes 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 101710096438 DNA-binding protein Proteins 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241001646716 Escherichia coli K-12 Species 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 102000006833 Multifunctional Enzymes Human genes 0.000 description 1
- 108010047290 Multifunctional Enzymes Proteins 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 1
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 238000012181 QIAquick gel extraction kit Methods 0.000 description 1
- 108010056079 Subtilisins Proteins 0.000 description 1
- 102000005158 Subtilisins Human genes 0.000 description 1
- 241000534944 Thia Species 0.000 description 1
- 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 1
- DLLXAZJTLIUPAI-XLPZGREQSA-N [[(2r,3s,5r)-5-(2-amino-4-oxo-1h-pyrrolo[2,3-d]pyrimidin-7-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound C1=2NC(N)=NC(=O)C=2C=CN1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 DLLXAZJTLIUPAI-XLPZGREQSA-N 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229960003237 betaine Drugs 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 239000007806 chemical reaction intermediate Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- UHZZMRAGKVHANO-UHFFFAOYSA-M chlormequat chloride Chemical compound [Cl-].C[N+](C)(C)CCCl UHZZMRAGKVHANO-UHFFFAOYSA-M 0.000 description 1
- 238000005094 computer simulation Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- UFJPAQSLHAGEBL-RRKCRQDMSA-N dITP Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(N=CNC2=O)=C2N=C1 UFJPAQSLHAGEBL-RRKCRQDMSA-N 0.000 description 1
- JSRLJPSBLDHEIO-SHYZEUOFSA-N dUMP Chemical compound O1[C@H](COP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 JSRLJPSBLDHEIO-SHYZEUOFSA-N 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000013461 design 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
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000009088 enzymatic function Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000003505 heat denaturation Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000003455 independent Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000010801 machine learning Methods 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 150000004712 monophosphates Chemical class 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 229920002113 octoxynol Polymers 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 230000001566 pro-viral effect Effects 0.000 description 1
- 230000001915 proofreading effect Effects 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 239000002719 pyrimidine nucleotide Substances 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000010079 rubber tapping Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 108010068698 spleen exonuclease Proteins 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005451 thionucleotide Substances 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
- C12N9/0032—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with oxygen as acceptor (1.5.3)
- C12N9/0034—Sarcosine oxidase (1.5.3.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/102—Mutagenizing nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Crystallography & Structural Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Enzymes And Modification Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP01115424 | 2001-06-27 | ||
| EP01115424.2 | 2001-06-27 | ||
| PCT/EP2002/007060 WO2003002736A2 (en) | 2001-06-27 | 2002-06-26 | A walk-through technique for in vitro recombination of polynucleotide sequences |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2453864A1 true CA2453864A1 (en) | 2003-01-09 |
Family
ID=8177828
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002453864A Abandoned CA2453864A1 (en) | 2001-06-27 | 2002-06-26 | A walk-through technique for in vitro recombination of polynucleotide sequences |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP1404825A2 (enExample) |
| JP (1) | JP3967319B2 (enExample) |
| CA (1) | CA2453864A1 (enExample) |
| WO (1) | WO2003002736A2 (enExample) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2464738A4 (en) * | 2009-08-12 | 2013-05-01 | Nugen Technologies Inc | METHODS, COMPOSITIONS, AND KITS FOR GENERATING NUCLEIC ACID PRODUCTS SUBSTANTIALLY FREE OF MATRIX NUCLEIC ACID |
| EP2769007B1 (en) | 2011-10-19 | 2016-12-07 | Nugen Technologies, Inc. | Compositions and methods for directional nucleic acid amplification and sequencing |
| WO2013112923A1 (en) | 2012-01-26 | 2013-08-01 | Nugen Technologies, Inc. | Compositions and methods for targeted nucleic acid sequence enrichment and high efficiency library generation |
| WO2013191775A2 (en) | 2012-06-18 | 2013-12-27 | Nugen Technologies, Inc. | Compositions and methods for negative selection of non-desired nucleic acid sequences |
| US20150011396A1 (en) | 2012-07-09 | 2015-01-08 | Benjamin G. Schroeder | Methods for creating directional bisulfite-converted nucleic acid libraries for next generation sequencing |
| WO2014144092A1 (en) | 2013-03-15 | 2014-09-18 | Nugen Technologies, Inc. | Sequential sequencing |
| WO2015073711A1 (en) | 2013-11-13 | 2015-05-21 | Nugen Technologies, Inc. | Compositions and methods for identification of a duplicate sequencing read |
| WO2015131107A1 (en) | 2014-02-28 | 2015-09-03 | Nugen Technologies, Inc. | Reduced representation bisulfite sequencing with diversity adaptors |
| US10102337B2 (en) | 2014-08-06 | 2018-10-16 | Nugen Technologies, Inc. | Digital measurements from targeted sequencing |
| US12492430B2 (en) | 2017-04-11 | 2025-12-09 | Tecan Genomics, Inc. | Library quantitation and qualification |
| US11099202B2 (en) | 2017-10-20 | 2021-08-24 | Tecan Genomics, Inc. | Reagent delivery system |
| IL276343B2 (en) * | 2018-01-29 | 2024-10-01 | St Jude Childrens Res Hospital Inc | Method for nucleic acid amplification |
| CN113275053A (zh) | 2020-02-03 | 2021-08-20 | 帝肯基因组学公司 | 试剂存储系统 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5605793A (en) * | 1994-02-17 | 1997-02-25 | Affymax Technologies N.V. | Methods for in vitro recombination |
-
2002
- 2002-06-26 JP JP2003509098A patent/JP3967319B2/ja not_active Expired - Fee Related
- 2002-06-26 CA CA002453864A patent/CA2453864A1/en not_active Abandoned
- 2002-06-26 EP EP02747439A patent/EP1404825A2/en not_active Withdrawn
- 2002-06-26 WO PCT/EP2002/007060 patent/WO2003002736A2/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| EP1404825A2 (en) | 2004-04-07 |
| JP2004533259A (ja) | 2004-11-04 |
| JP3967319B2 (ja) | 2007-08-29 |
| WO2003002736A3 (en) | 2003-07-31 |
| WO2003002736A2 (en) | 2003-01-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US6177263B1 (en) | Recombination of polynucleotide sequences using random or defined primers | |
| US7833759B2 (en) | Method of increasing complementarity in a heteroduplex | |
| CA2453864A1 (en) | A walk-through technique for in vitro recombination of polynucleotide sequences | |
| AU2002314712A1 (en) | A method of increasing complementarity in a heteroduplex polynucleotide | |
| AU2002321251B2 (en) | Method for the production of nucleic acids consisting of stochastically combined parts of source nucleic acids | |
| US6361988B1 (en) | ECB deacylase mutants | |
| US6955879B2 (en) | Method for generating recombinant DNA library using unidirectional single-stranded DNA fragments | |
| US6994963B1 (en) | Methods for recombinatorial nucleic acid synthesis | |
| KR100571937B1 (ko) | 돌연변이 티로신 페놀리아제 및 이의 제조방법 | |
| US20030087254A1 (en) | Methods for the preparation of polynucleotide libraries and identification of library members having desired characteristics | |
| ES2280891T3 (es) | Metodo para obtener polinucleotidos circulares mutados y/o quimericos. | |
| HUP9903760A2 (hu) | Polinukleotid-szekvenciák rekombinációja véletlenszerű vagy meghatározott láncindítók alkalmazásával | |
| Shao et al. | Random-priming in vitro recombination: an effective | |
| MXPA98009854A (es) | Recombinacion de secuencias de polinucleotidos utilizando cebadores aleatorios o definidos | |
| AU7257300A (en) | Recombination of polynucleotide sequences using random or defined primers | |
| ZA200306203B (en) | A method of increasing complementarity in a heteroduplex polynucleotide. | |
| KR20000015984A (ko) | 무작위 프라이머 또는 일정 프라이머를 이용한폴리뉴클레오티드 서열의 재조합 방법 | |
| AU2002307170A1 (en) | Methods for the preparation of polynucleotide librairies and identification of library members having desired characteristics | |
| ZA200308639B (en) | Methods for the preparation of polynucleotide libraries and indentification of library members having desired characteristics. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |