JP5394928B2 - サーマス・エガートソニイ(thermuseggertssonii)dnaポリメラーゼ - Google Patents
サーマス・エガートソニイ(thermuseggertssonii)dnaポリメラーゼ Download PDFInfo
- Publication number
- JP5394928B2 JP5394928B2 JP2009531829A JP2009531829A JP5394928B2 JP 5394928 B2 JP5394928 B2 JP 5394928B2 JP 2009531829 A JP2009531829 A JP 2009531829A JP 2009531829 A JP2009531829 A JP 2009531829A JP 5394928 B2 JP5394928 B2 JP 5394928B2
- Authority
- JP
- Japan
- Prior art keywords
- dna
- polymerase
- teg
- nucleic acid
- dna polymerase
- 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.)
- Expired - Fee Related
Links
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 title claims abstract description 164
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 title claims abstract description 164
- 241001602803 Thermus eggertssonii Species 0.000 title description 31
- 150000007523 nucleic acids Chemical group 0.000 claims abstract description 224
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 181
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 181
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 119
- 239000013598 vector Substances 0.000 claims abstract description 35
- 238000003556 assay Methods 0.000 claims abstract description 27
- 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 claims abstract description 9
- 230000004543 DNA replication Effects 0.000 claims abstract description 9
- 238000011534 incubation Methods 0.000 claims abstract description 9
- 238000000338 in vitro Methods 0.000 claims abstract description 8
- 102000004594 DNA Polymerase I Human genes 0.000 claims description 196
- 108010017826 DNA Polymerase I Proteins 0.000 claims description 190
- 108090000623 proteins and genes Proteins 0.000 claims description 126
- 108020004414 DNA Proteins 0.000 claims description 115
- 125000003729 nucleotide group Chemical group 0.000 claims description 46
- 239000002773 nucleotide Substances 0.000 claims description 44
- 230000010076 replication Effects 0.000 claims description 41
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 40
- 230000027455 binding Effects 0.000 claims description 22
- 238000012197 amplification kit Methods 0.000 claims description 15
- 239000003550 marker Substances 0.000 claims description 12
- 230000004544 DNA amplification Effects 0.000 claims description 9
- 238000001712 DNA sequencing Methods 0.000 claims description 9
- 239000011535 reaction buffer Substances 0.000 claims description 7
- 238000013519 translation Methods 0.000 claims description 5
- 108700039691 Genetic Promoter Regions Proteins 0.000 claims description 4
- 230000002503 metabolic effect Effects 0.000 claims description 2
- 108091027963 non-coding RNA Proteins 0.000 claims description 2
- 102000042567 non-coding RNA Human genes 0.000 claims description 2
- 230000036961 partial effect Effects 0.000 abstract description 13
- 239000013615 primer Substances 0.000 description 129
- 238000003752 polymerase chain reaction Methods 0.000 description 107
- 238000000034 method Methods 0.000 description 96
- 102000004169 proteins and genes Human genes 0.000 description 95
- 210000004027 cell Anatomy 0.000 description 94
- 238000006243 chemical reaction Methods 0.000 description 90
- 235000018102 proteins Nutrition 0.000 description 87
- 241000589596 Thermus Species 0.000 description 69
- 230000003321 amplification Effects 0.000 description 69
- 238000003199 nucleic acid amplification method Methods 0.000 description 69
- 239000000047 product Substances 0.000 description 66
- 230000000694 effects Effects 0.000 description 55
- 239000011541 reaction mixture Substances 0.000 description 48
- 108090000765 processed proteins & peptides Proteins 0.000 description 43
- 102000004190 Enzymes Human genes 0.000 description 40
- 108090000790 Enzymes Proteins 0.000 description 40
- 229940088598 enzyme Drugs 0.000 description 40
- 102000004196 processed proteins & peptides Human genes 0.000 description 37
- 238000004458 analytical method Methods 0.000 description 36
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 35
- 108010006785 Taq Polymerase Proteins 0.000 description 34
- 235000001014 amino acid Nutrition 0.000 description 34
- 101150055096 polA gene Proteins 0.000 description 33
- 108060002716 Exonuclease Proteins 0.000 description 32
- 102000013165 exonuclease Human genes 0.000 description 32
- 239000013604 expression vector Substances 0.000 description 32
- 229920001184 polypeptide Polymers 0.000 description 32
- 238000012163 sequencing technique Methods 0.000 description 30
- 229940024606 amino acid Drugs 0.000 description 28
- 150000001413 amino acids Chemical group 0.000 description 28
- 241000894007 species Species 0.000 description 27
- 241000894006 Bacteria Species 0.000 description 26
- 239000012634 fragment Substances 0.000 description 26
- 238000006467 substitution reaction Methods 0.000 description 26
- 239000000872 buffer Substances 0.000 description 25
- 239000000758 substrate Substances 0.000 description 25
- 101100064044 Schizosaccharomyces pombe (strain 972 / ATCC 24843) pol1 gene Proteins 0.000 description 23
- 108020001507 fusion proteins Proteins 0.000 description 23
- 102000037865 fusion proteins Human genes 0.000 description 23
- 101150088264 pol gene Proteins 0.000 description 23
- 238000000746 purification Methods 0.000 description 23
- 241000589499 Thermus thermophilus Species 0.000 description 22
- 102000053602 DNA Human genes 0.000 description 21
- 241000588724 Escherichia coli Species 0.000 description 21
- 230000000295 complement effect Effects 0.000 description 21
- 239000000499 gel Substances 0.000 description 19
- 239000000203 mixture Substances 0.000 description 19
- 239000000523 sample Substances 0.000 description 19
- 239000000284 extract Substances 0.000 description 18
- 230000002068 genetic effect Effects 0.000 description 17
- 239000001103 potassium chloride Substances 0.000 description 17
- 235000011164 potassium chloride Nutrition 0.000 description 17
- 238000003753 real-time PCR Methods 0.000 description 17
- 238000012360 testing method Methods 0.000 description 17
- 108091026890 Coding region Proteins 0.000 description 16
- 241000589500 Thermus aquaticus Species 0.000 description 16
- 239000002609 medium Substances 0.000 description 16
- 239000002253 acid Substances 0.000 description 15
- 230000001580 bacterial effect Effects 0.000 description 15
- 238000010790 dilution Methods 0.000 description 15
- 239000012895 dilution Substances 0.000 description 15
- 230000012010 growth Effects 0.000 description 15
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 14
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 14
- 230000015572 biosynthetic process Effects 0.000 description 14
- 238000001962 electrophoresis Methods 0.000 description 14
- 238000010348 incorporation Methods 0.000 description 14
- 108020004465 16S ribosomal RNA Proteins 0.000 description 13
- -1 Nucleoside triphosphates Chemical class 0.000 description 13
- 238000002474 experimental method Methods 0.000 description 13
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 13
- 238000003786 synthesis reaction Methods 0.000 description 13
- 108700028369 Alleles Proteins 0.000 description 12
- 108020004705 Codon Proteins 0.000 description 12
- 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 12
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 12
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 12
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 12
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 12
- 210000003811 finger Anatomy 0.000 description 12
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 12
- 210000003813 thumb Anatomy 0.000 description 12
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 11
- 235000013922 glutamic acid Nutrition 0.000 description 11
- 239000004220 glutamic acid Substances 0.000 description 11
- 239000013612 plasmid Substances 0.000 description 11
- 230000001105 regulatory effect Effects 0.000 description 11
- 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 10
- 239000004472 Lysine Substances 0.000 description 10
- 108091034117 Oligonucleotide Proteins 0.000 description 10
- 239000007983 Tris buffer Substances 0.000 description 10
- 238000012217 deletion Methods 0.000 description 10
- 230000037430 deletion Effects 0.000 description 10
- 230000001965 increasing effect Effects 0.000 description 10
- 230000006698 induction Effects 0.000 description 10
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 10
- UEGPKNKPLBYCNK-UHFFFAOYSA-L magnesium acetate Chemical compound [Mg+2].CC([O-])=O.CC([O-])=O UEGPKNKPLBYCNK-UHFFFAOYSA-L 0.000 description 10
- 239000011654 magnesium acetate Substances 0.000 description 10
- 235000011285 magnesium acetate Nutrition 0.000 description 10
- 229940069446 magnesium acetate Drugs 0.000 description 10
- 230000004048 modification Effects 0.000 description 10
- 238000012986 modification Methods 0.000 description 10
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 10
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 9
- 229920001817 Agar Polymers 0.000 description 9
- 241000589498 Thermus filiformis Species 0.000 description 9
- 239000008272 agar Substances 0.000 description 9
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 9
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 9
- 235000011130 ammonium sulphate Nutrition 0.000 description 9
- 230000004927 fusion Effects 0.000 description 9
- 230000037452 priming Effects 0.000 description 9
- 230000002829 reductive effect Effects 0.000 description 9
- 238000002864 sequence alignment Methods 0.000 description 9
- 239000012619 Butyl Sepharose® Substances 0.000 description 8
- FSVCELGFZIQNCK-UHFFFAOYSA-N N,N-bis(2-hydroxyethyl)glycine Chemical compound OCCN(CCO)CC(O)=O FSVCELGFZIQNCK-UHFFFAOYSA-N 0.000 description 8
- 150000007513 acids Chemical class 0.000 description 8
- 239000007998 bicine buffer Substances 0.000 description 8
- 239000003153 chemical reaction reagent Substances 0.000 description 8
- 150000001875 compounds Chemical class 0.000 description 8
- 239000006167 equilibration buffer Substances 0.000 description 8
- 238000009396 hybridization Methods 0.000 description 8
- 230000001976 improved effect Effects 0.000 description 8
- 238000003780 insertion Methods 0.000 description 8
- 230000037431 insertion Effects 0.000 description 8
- 230000035772 mutation Effects 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 8
- 238000013518 transcription Methods 0.000 description 8
- 230000035897 transcription Effects 0.000 description 8
- 102000000662 3'-5' exonuclease domains Human genes 0.000 description 7
- 108050008023 3'-5' exonuclease domains Proteins 0.000 description 7
- 239000004475 Arginine Substances 0.000 description 7
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 7
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 7
- 229920002873 Polyethylenimine Polymers 0.000 description 7
- 229920002684 Sepharose Polymers 0.000 description 7
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 7
- 235000009697 arginine Nutrition 0.000 description 7
- 210000004899 c-terminal region Anatomy 0.000 description 7
- 238000010367 cloning Methods 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 230000006870 function Effects 0.000 description 7
- 229960002897 heparin Drugs 0.000 description 7
- 229920000669 heparin Polymers 0.000 description 7
- 229910001425 magnesium ion Inorganic materials 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 241000238631 Hexapoda Species 0.000 description 6
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 6
- 238000009004 PCR Kit Methods 0.000 description 6
- 229960000723 ampicillin Drugs 0.000 description 6
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 6
- 238000000137 annealing Methods 0.000 description 6
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 239000011777 magnesium Substances 0.000 description 6
- 229910052749 magnesium Inorganic materials 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 102000040430 polynucleotide Human genes 0.000 description 6
- 108091033319 polynucleotide Proteins 0.000 description 6
- 239000002157 polynucleotide Substances 0.000 description 6
- 239000002987 primer (paints) Substances 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 230000002103 transcriptional effect Effects 0.000 description 6
- 238000011144 upstream manufacturing Methods 0.000 description 6
- 241000193830 Bacillus <bacterium> Species 0.000 description 5
- 239000003155 DNA primer Substances 0.000 description 5
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 5
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 5
- 125000000174 L-prolyl group Chemical group [H]N1C([H])([H])C([H])([H])C([H])([H])[C@@]1([H])C(*)=O 0.000 description 5
- 101710163270 Nuclease Proteins 0.000 description 5
- 239000004743 Polypropylene Substances 0.000 description 5
- 108010076504 Protein Sorting Signals Proteins 0.000 description 5
- 108020004682 Single-Stranded DNA Proteins 0.000 description 5
- 241000557726 Thermus oshimai Species 0.000 description 5
- 239000011543 agarose gel Substances 0.000 description 5
- 125000000539 amino acid group Chemical group 0.000 description 5
- 235000003704 aspartic acid Nutrition 0.000 description 5
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 5
- 229910052799 carbon Inorganic materials 0.000 description 5
- 239000007795 chemical reaction product Substances 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
- 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 5
- 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 5
- 238000004925 denaturation Methods 0.000 description 5
- 230000036425 denaturation Effects 0.000 description 5
- 239000012149 elution buffer Substances 0.000 description 5
- 238000007848 endpoint PCR Methods 0.000 description 5
- 239000003623 enhancer Substances 0.000 description 5
- 238000010438 heat treatment Methods 0.000 description 5
- 230000001939 inductive effect Effects 0.000 description 5
- 230000007246 mechanism Effects 0.000 description 5
- 238000002703 mutagenesis Methods 0.000 description 5
- 231100000350 mutagenesis Toxicity 0.000 description 5
- 239000002777 nucleoside Substances 0.000 description 5
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 5
- 229920001155 polypropylene Polymers 0.000 description 5
- 150000003839 salts Chemical class 0.000 description 5
- 230000028327 secretion Effects 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 4
- 230000004568 DNA-binding Effects 0.000 description 4
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 4
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 4
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 4
- 125000002059 L-arginyl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])C([H])([H])C([H])([H])N([H])C(=N[H])N([H])[H] 0.000 description 4
- 108060004795 Methyltransferase Proteins 0.000 description 4
- 125000000729 N-terminal amino-acid group Chemical group 0.000 description 4
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 241000205188 Thermococcus Species 0.000 description 4
- 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 4
- 230000000692 anti-sense effect Effects 0.000 description 4
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 4
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 4
- 230000004071 biological effect Effects 0.000 description 4
- 230000001419 dependent effect Effects 0.000 description 4
- 239000012636 effector Substances 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 230000000670 limiting effect Effects 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 210000004962 mammalian cell Anatomy 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 230000003647 oxidation Effects 0.000 description 4
- 238000007254 oxidation reaction Methods 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 4
- 230000008488 polyadenylation Effects 0.000 description 4
- 238000011002 quantification Methods 0.000 description 4
- 230000002441 reversible effect Effects 0.000 description 4
- 239000000600 sorbitol Substances 0.000 description 4
- 239000003381 stabilizer Substances 0.000 description 4
- 230000000087 stabilizing effect Effects 0.000 description 4
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 4
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 4
- 241000203069 Archaea Species 0.000 description 3
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 3
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 3
- 239000002028 Biomass Substances 0.000 description 3
- 108010071146 DNA Polymerase III Proteins 0.000 description 3
- 102000007528 DNA Polymerase III Human genes 0.000 description 3
- 230000006820 DNA synthesis Effects 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 3
- 102000005548 Hexokinase Human genes 0.000 description 3
- 108700040460 Hexokinases Proteins 0.000 description 3
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 3
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 3
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 3
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 3
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 3
- 108091005461 Nucleic proteins Chemical group 0.000 description 3
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 3
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 108700026226 TATA Box Proteins 0.000 description 3
- 239000004098 Tetracycline Substances 0.000 description 3
- 108700009124 Transcription Initiation Site Proteins 0.000 description 3
- 238000001042 affinity chromatography Methods 0.000 description 3
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 150000001720 carbohydrates Chemical class 0.000 description 3
- 235000014633 carbohydrates Nutrition 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 229960005091 chloramphenicol Drugs 0.000 description 3
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 230000001351 cycling effect Effects 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- 229930182830 galactose Natural products 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000002779 inactivation Effects 0.000 description 3
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000010369 molecular cloning Methods 0.000 description 3
- 238000002135 phase contrast microscopy Methods 0.000 description 3
- 229920002401 polyacrylamide Polymers 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 238000006116 polymerization reaction Methods 0.000 description 3
- 229910001414 potassium ion Inorganic materials 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 230000001915 proofreading effect Effects 0.000 description 3
- 230000002285 radioactive effect Effects 0.000 description 3
- 238000002708 random mutagenesis Methods 0.000 description 3
- 230000008439 repair process Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- DHCDFWKWKRSZHF-UHFFFAOYSA-N sulfurothioic S-acid Chemical compound OS(O)(=O)=S DHCDFWKWKRSZHF-UHFFFAOYSA-N 0.000 description 3
- 229960002180 tetracycline Drugs 0.000 description 3
- 229930101283 tetracycline Natural products 0.000 description 3
- 235000019364 tetracycline Nutrition 0.000 description 3
- 150000003522 tetracyclines Chemical class 0.000 description 3
- 238000005382 thermal cycling Methods 0.000 description 3
- 230000005026 transcription initiation Effects 0.000 description 3
- 239000001226 triphosphate Substances 0.000 description 3
- 235000011178 triphosphate Nutrition 0.000 description 3
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 2
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- OQRXBXNATIHDQO-UHFFFAOYSA-N 6-chloropyridine-3,4-diamine Chemical compound NC1=CN=C(Cl)C=C1N OQRXBXNATIHDQO-UHFFFAOYSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 description 2
- 108020004513 Bacterial RNA Proteins 0.000 description 2
- 102100035882 Catalase Human genes 0.000 description 2
- 108010053835 Catalase Proteins 0.000 description 2
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 2
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- 108010063113 DNA Polymerase II Proteins 0.000 description 2
- 102000010567 DNA Polymerase II Human genes 0.000 description 2
- 230000005778 DNA damage Effects 0.000 description 2
- 231100000277 DNA damage Toxicity 0.000 description 2
- 102000004214 DNA polymerase A Human genes 0.000 description 2
- 108090000725 DNA polymerase A Proteins 0.000 description 2
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 2
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 2
- 101900234631 Escherichia coli DNA polymerase I Proteins 0.000 description 2
- 108090000371 Esterases Proteins 0.000 description 2
- 241000282326 Felis catus Species 0.000 description 2
- 229930182566 Gentamicin Natural products 0.000 description 2
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 229930010555 Inosine Natural products 0.000 description 2
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 2
- 102000012011 Isocitrate Dehydrogenase Human genes 0.000 description 2
- 108010075869 Isocitrate Dehydrogenase Proteins 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 2
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- 102000013460 Malate Dehydrogenase Human genes 0.000 description 2
- 108010026217 Malate Dehydrogenase Proteins 0.000 description 2
- 108091092878 Microsatellite Proteins 0.000 description 2
- 101100172630 Mus musculus Eri1 gene Proteins 0.000 description 2
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 2
- 229910002651 NO3 Inorganic materials 0.000 description 2
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 238000002944 PCR assay Methods 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 241000235648 Pichia Species 0.000 description 2
- 239000004698 Polyethylene Substances 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 102100036286 Purine nucleoside phosphorylase Human genes 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- 108091028733 RNTP Proteins 0.000 description 2
- 102000001218 Rec A Recombinases Human genes 0.000 description 2
- 108010055016 Rec A Recombinases Proteins 0.000 description 2
- 108091028664 Ribonucleotide Proteins 0.000 description 2
- 241000700584 Simplexvirus Species 0.000 description 2
- FKNQFGJONOIPTF-UHFFFAOYSA-N Sodium cation Chemical compound [Na+] FKNQFGJONOIPTF-UHFFFAOYSA-N 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 108091081024 Start codon Proteins 0.000 description 2
- 241000194017 Streptococcus Species 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 102000019197 Superoxide Dismutase Human genes 0.000 description 2
- 108010012715 Superoxide dismutase Proteins 0.000 description 2
- 241000204652 Thermotoga Species 0.000 description 2
- 241000868182 Thermus thermophilus HB8 Species 0.000 description 2
- 241000605118 Thiobacillus Species 0.000 description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- PPQRONHOSHZGFQ-LMVFSUKVSA-N aldehydo-D-ribose 5-phosphate Chemical group OP(=O)(O)OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PPQRONHOSHZGFQ-LMVFSUKVSA-N 0.000 description 2
- 238000007844 allele-specific PCR Methods 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 2
- 230000001588 bifunctional effect Effects 0.000 description 2
- 230000001851 biosynthetic effect Effects 0.000 description 2
- 108091005948 blue fluorescent proteins Proteins 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000010307 cell transformation Effects 0.000 description 2
- JQXXHWHPUNPDRT-YOPQJBRCSA-N chembl1332716 Chemical compound O([C@](C1=O)(C)O\C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)/C=C\C=C(C)/C(=O)NC=2C(O)=C3C(O)=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CCN(C)CC1 JQXXHWHPUNPDRT-YOPQJBRCSA-N 0.000 description 2
- 238000013375 chromatographic separation Methods 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 230000001143 conditioned effect Effects 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 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 2
- 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 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000005546 dideoxynucleotide Substances 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 229960002518 gentamicin Drugs 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- 239000011544 gradient gel Substances 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 229960003786 inosine Drugs 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 230000002427 irreversible effect Effects 0.000 description 2
- DRAVOWXCEBXPTN-UHFFFAOYSA-N isoguanine Chemical compound NC1=NC(=O)NC2=C1NC=N2 DRAVOWXCEBXPTN-UHFFFAOYSA-N 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 239000012160 loading buffer Substances 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- MHWLWQUZZRMNGJ-UHFFFAOYSA-N nalidixic acid Chemical compound C1=C(C)N=C2N(CC)C=C(C(O)=O)C(=O)C2=C1 MHWLWQUZZRMNGJ-UHFFFAOYSA-N 0.000 description 2
- 229960000210 nalidixic acid Drugs 0.000 description 2
- 238000007826 nucleic acid assay Methods 0.000 description 2
- 125000003835 nucleoside group Chemical group 0.000 description 2
- 108010009099 nucleoside phosphorylase Proteins 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 150000004713 phosphodiesters Chemical class 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 102000054765 polymorphisms of proteins Human genes 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 125000001500 prolyl group Chemical group [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 description 2
- 235000019833 protease Nutrition 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- 238000010188 recombinant method Methods 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 108010054624 red fluorescent protein Proteins 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000003362 replicative effect Effects 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- 239000002336 ribonucleotide Substances 0.000 description 2
- 125000002652 ribonucleotide group Chemical group 0.000 description 2
- 229960001225 rifampicin Drugs 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 229910001415 sodium ion Inorganic materials 0.000 description 2
- 238000010532 solid phase synthesis reaction Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000012536 storage buffer Substances 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000005030 transcription termination Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 241000701447 unidentified baculovirus Species 0.000 description 2
- 241001515965 unidentified phage Species 0.000 description 2
- 108010064245 urinary gonadotropin fragment Proteins 0.000 description 2
- 238000011179 visual inspection Methods 0.000 description 2
- 108091005957 yellow fluorescent proteins Proteins 0.000 description 2
- FXYPGCIGRDZWNR-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-[[3-(2,5-dioxopyrrolidin-1-yl)oxy-3-oxopropyl]disulfanyl]propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCSSCCC(=O)ON1C(=O)CCC1=O FXYPGCIGRDZWNR-UHFFFAOYSA-N 0.000 description 1
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- VYEWZWBILJHHCU-OMQUDAQFSA-N (e)-n-[(2s,3r,4r,5r,6r)-2-[(2r,3r,4s,5s,6s)-3-acetamido-5-amino-4-hydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-6-[2-[(2r,3s,4r,5r)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]-2-hydroxyethyl]-4,5-dihydroxyoxan-3-yl]-5-methylhex-2-enamide Chemical compound N1([C@@H]2O[C@@H]([C@H]([C@H]2O)O)C(O)C[C@@H]2[C@H](O)[C@H](O)[C@H]([C@@H](O2)O[C@@H]2[C@@H]([C@@H](O)[C@H](N)[C@@H](CO)O2)NC(C)=O)NC(=O)/C=C/CC(C)C)C=CC(=O)NC1=O VYEWZWBILJHHCU-OMQUDAQFSA-N 0.000 description 1
- CJAOGUFAAWZWNI-UHFFFAOYSA-N 1-n,1-n,4-n,4-n-tetramethylbenzene-1,4-diamine Chemical compound CN(C)C1=CC=C(N(C)C)C=C1 CJAOGUFAAWZWNI-UHFFFAOYSA-N 0.000 description 1
- 125000003287 1H-imidazol-4-ylmethyl group Chemical group [H]N1C([H])=NC(C([H])([H])[*])=C1[H] 0.000 description 1
- 150000003923 2,5-pyrrolediones Chemical class 0.000 description 1
- XQCZBXHVTFVIFE-UHFFFAOYSA-N 2-amino-4-hydroxypyrimidine Chemical compound NC1=NC=CC(O)=N1 XQCZBXHVTFVIFE-UHFFFAOYSA-N 0.000 description 1
- BIGBDMFRWJRLGJ-UHFFFAOYSA-N 3-benzyl-1,5-didiazoniopenta-1,4-diene-2,4-diolate Chemical compound [N-]=[N+]=CC(=O)C(C(=O)C=[N+]=[N-])CC1=CC=CC=C1 BIGBDMFRWJRLGJ-UHFFFAOYSA-N 0.000 description 1
- OSJPPGNTCRNQQC-UWTATZPHSA-N 3-phospho-D-glyceric acid Chemical compound OC(=O)[C@H](O)COP(O)(O)=O OSJPPGNTCRNQQC-UWTATZPHSA-N 0.000 description 1
- NLPWSMKACWGINL-UHFFFAOYSA-N 4-azido-2-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(N=[N+]=[N-])C=C1O NLPWSMKACWGINL-UHFFFAOYSA-N 0.000 description 1
- 108010051457 Acid Phosphatase Proteins 0.000 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- 102100034613 Annexin A2 Human genes 0.000 description 1
- 108010053481 Antifreeze Proteins Proteins 0.000 description 1
- 241000207208 Aquifex Species 0.000 description 1
- 241000319477 Asellus aquaticus Species 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 102100022717 Atypical chemokine receptor 1 Human genes 0.000 description 1
- 108010001478 Bacitracin Proteins 0.000 description 1
- 244000177578 Bacterium linens Species 0.000 description 1
- SGHZXLIDFTYFHQ-UHFFFAOYSA-L Brilliant Blue Chemical compound [Na+].[Na+].C=1C=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C(=CC=CC=2)S([O-])(=O)=O)C=CC=1N(CC)CC1=CC=CC(S([O-])(=O)=O)=C1 SGHZXLIDFTYFHQ-UHFFFAOYSA-L 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- 101100327917 Caenorhabditis elegans chup-1 gene Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 101000909256 Caldicellulosiruptor bescii (strain ATCC BAA-1888 / DSM 6725 / Z-1320) DNA polymerase I Proteins 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 241001112695 Clostridiales Species 0.000 description 1
- 241000193403 Clostridium Species 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 1
- 241000192700 Cyanobacteria Species 0.000 description 1
- 102000003915 DNA Topoisomerases Human genes 0.000 description 1
- 108090000323 DNA Topoisomerases Proteins 0.000 description 1
- 230000033616 DNA repair Effects 0.000 description 1
- 101710141836 DNA-binding protein HU homolog Proteins 0.000 description 1
- 101710088194 Dehydrogenase 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
- 241000186541 Desulfotomaculum Species 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 108090000204 Dipeptidase 1 Proteins 0.000 description 1
- 241000255601 Drosophila melanogaster Species 0.000 description 1
- 108010091873 DyNAzyme polymerase Proteins 0.000 description 1
- 241000544050 Egeria Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 101710202200 Endolysin A Proteins 0.000 description 1
- 102100031780 Endonuclease Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 241001524679 Escherichia virus M13 Species 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000626621 Geobacillus Species 0.000 description 1
- 108010021582 Glucokinase Proteins 0.000 description 1
- 102000030595 Glucokinase Human genes 0.000 description 1
- 108010070600 Glucose-6-phosphate isomerase Proteins 0.000 description 1
- 102000005731 Glucose-6-phosphate isomerase Human genes 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 241000707503 Greeneria Species 0.000 description 1
- HVLSXIKZNLPZJJ-TXZCQADKSA-N HA peptide Chemical compound C([C@@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 HVLSXIKZNLPZJJ-TXZCQADKSA-N 0.000 description 1
- 101150069554 HIS4 gene Proteins 0.000 description 1
- 108091005904 Hemoglobin subunit beta Proteins 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- LYCVKHSJGDMDLM-LURJTMIESA-N His-Gly Chemical compound OC(=O)CNC(=O)[C@@H](N)CC1=CN=CN1 LYCVKHSJGDMDLM-LURJTMIESA-N 0.000 description 1
- 101000756632 Homo sapiens Actin, cytoplasmic 1 Proteins 0.000 description 1
- 101000678879 Homo sapiens Atypical chemokine receptor 1 Proteins 0.000 description 1
- 101001033034 Homo sapiens UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase Proteins 0.000 description 1
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 102000004195 Isomerases Human genes 0.000 description 1
- 108090000769 Isomerases Proteins 0.000 description 1
- 108010025815 Kanamycin Kinase Proteins 0.000 description 1
- 244000285963 Kluyveromyces fragilis Species 0.000 description 1
- 235000014663 Kluyveromyces fragilis Nutrition 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- 125000000393 L-methionino group Chemical group [H]OC(=O)[C@@]([H])(N([H])[*])C([H])([H])C(SC([H])([H])[H])([H])[H] 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 241000194034 Lactococcus lactis subsp. cremoris Species 0.000 description 1
- 108091027974 Mature messenger RNA Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000203353 Methanococcus Species 0.000 description 1
- 101710174628 Modulating protein YmoA Proteins 0.000 description 1
- 241000713333 Mouse mammary tumor virus Species 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010021466 Mutant Proteins Proteins 0.000 description 1
- 102000008300 Mutant Proteins Human genes 0.000 description 1
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical class ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 241000221960 Neurospora Species 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- 241000320412 Ogataea angusta Species 0.000 description 1
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 1
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 108091093037 Peptide nucleic acid Proteins 0.000 description 1
- 108010022181 Phosphopyruvate Hydratase Proteins 0.000 description 1
- 102000012288 Phosphopyruvate Hydratase Human genes 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 108010015078 Pregnancy-Associated alpha 2-Macroglobulins Proteins 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- KDCGOANMDULRCW-UHFFFAOYSA-N Purine Natural products N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 1
- 241000205160 Pyrococcus Species 0.000 description 1
- 101000902592 Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1) DNA polymerase Proteins 0.000 description 1
- 102000009609 Pyrophosphatases Human genes 0.000 description 1
- 108010009413 Pyrophosphatases Proteins 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- 102000013009 Pyruvate Kinase Human genes 0.000 description 1
- 102000009572 RNA Polymerase II Human genes 0.000 description 1
- 108010009460 RNA Polymerase II Proteins 0.000 description 1
- 230000006819 RNA synthesis Effects 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 230000027151 SOS response Effects 0.000 description 1
- 241000235070 Saccharomyces Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 241000235347 Schizosaccharomyces pombe Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 241000589970 Spirochaetales Species 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 235000014962 Streptococcus cremoris Nutrition 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 1
- 241000205101 Sulfolobus Species 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- 241000959851 Thermales Species 0.000 description 1
- 241000204667 Thermoplasma Species 0.000 description 1
- 241000204673 Thermoplasma acidophilum Species 0.000 description 1
- 241000204666 Thermotoga maritima Species 0.000 description 1
- 241000204664 Thermotoga neapolitana Species 0.000 description 1
- 241000557720 Thermus brockianus Species 0.000 description 1
- 241001522143 Thermus scotoductus Species 0.000 description 1
- 241000589497 Thermus sp. Species 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 108010075344 Tryptophan synthase Proteins 0.000 description 1
- 102000004243 Tubulin Human genes 0.000 description 1
- 108090000704 Tubulin Proteins 0.000 description 1
- YJQCOFNZVFGCAF-UHFFFAOYSA-N Tunicamycin II Natural products O1C(CC(O)C2C(C(O)C(O2)N2C(NC(=O)C=C2)=O)O)C(O)C(O)C(NC(=O)C=CCCCCCCCCC(C)C)C1OC1OC(CO)C(O)C(O)C1NC(C)=O YJQCOFNZVFGCAF-UHFFFAOYSA-N 0.000 description 1
- 102100038413 UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase Human genes 0.000 description 1
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 108010059993 Vancomycin Proteins 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 241000235015 Yarrowia lipolytica Species 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 239000000061 acid fraction Substances 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 208000005652 acute fatty liver of pregnancy Diseases 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 1
- HWXBTNAVRSUOJR-UHFFFAOYSA-N alpha-hydroxyglutaric acid Natural products OC(=O)C(O)CCC(O)=O HWXBTNAVRSUOJR-UHFFFAOYSA-N 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 229940009533 alpha-ketoglutaric acid Drugs 0.000 description 1
- 230000009435 amidation Effects 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 150000001412 amines Chemical group 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 description 1
- 229960003071 bacitracin Drugs 0.000 description 1
- 229930184125 bacitracin Natural products 0.000 description 1
- CLKOFPXJLQSYAH-ABRJDSQDSA-N bacitracin A Chemical compound C1SC([C@@H](N)[C@@H](C)CC)=N[C@@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]1C(=O)N[C@H](CCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2N=CNC=2)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCCCC1 CLKOFPXJLQSYAH-ABRJDSQDSA-N 0.000 description 1
- 102000006635 beta-lactamase Human genes 0.000 description 1
- 238000011953 bioanalysis Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 210000004252 chorionic villi Anatomy 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 235000015165 citric acid Nutrition 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229910001431 copper ion Inorganic materials 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 239000006184 cosolvent Substances 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000006240 deamidation Effects 0.000 description 1
- 238000000326 densiometry Methods 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000000368 destabilizing effect Effects 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- NAGJZTKCGNOGPW-UHFFFAOYSA-K dioxido-sulfanylidene-sulfido-$l^{5}-phosphane Chemical compound [O-]P([O-])([S-])=S NAGJZTKCGNOGPW-UHFFFAOYSA-K 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 125000004119 disulfanediyl group Chemical group *SS* 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229960003276 erythromycin Drugs 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 238000013100 final test Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000037433 frameshift Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 description 1
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 238000003505 heat denaturation Methods 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000033444 hydroxylation Effects 0.000 description 1
- 238000005805 hydroxylation reaction Methods 0.000 description 1
- 230000015784 hyperosmotic salinity response Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000009830 intercalation Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000001155 isoelectric focusing Methods 0.000 description 1
- 125000000741 isoleucyl group Chemical group [H]N([H])C(C(C([H])([H])[H])C([H])([H])C([H])([H])[H])C(=O)O* 0.000 description 1
- 229950003188 isovaleryl diethylamide Drugs 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 238000012886 linear function Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 108010026228 mRNA guanylyltransferase Proteins 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- RXMBKOPBFXCPDD-UHFFFAOYSA-N methoxyphosphonamidous acid Chemical compound COP(N)O RXMBKOPBFXCPDD-UHFFFAOYSA-N 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000002887 multiple sequence alignment Methods 0.000 description 1
- 210000000066 myeloid cell Anatomy 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 208000007538 neurilemmoma Diseases 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 229960003104 ornithine Drugs 0.000 description 1
- 229940056360 penicillin g Drugs 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 210000001322 periplasm Anatomy 0.000 description 1
- 125000000405 phenylalanyl group Chemical group 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 150000008039 phosphoramides Chemical class 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000000243 photosynthetic effect Effects 0.000 description 1
- 238000013081 phylogenetic analysis Methods 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 238000007692 polyacrylamide-agarose gel electrophoresis Methods 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920002704 polyhistidine Polymers 0.000 description 1
- 229920001451 polypropylene glycol Polymers 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 238000011533 pre-incubation Methods 0.000 description 1
- 238000002953 preparative HPLC Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- QLNJFJADRCOGBJ-UHFFFAOYSA-N propionamide Chemical compound CCC(N)=O QLNJFJADRCOGBJ-UHFFFAOYSA-N 0.000 description 1
- 230000004952 protein activity Effects 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 125000000561 purinyl group Chemical group N1=C(N=C2N=CNC2=C1)* 0.000 description 1
- 125000000714 pyrimidinyl group Chemical group 0.000 description 1
- 229940076788 pyruvate Drugs 0.000 description 1
- 108010010870 pyruvate kinase phosphatase Proteins 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000010807 real-time PCR kit Methods 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 150000003290 ribose derivatives Chemical group 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000007480 sanger sequencing Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 206010039667 schwannoma Diseases 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000007423 screening assay Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000012134 supernatant fraction Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 1
- 235000008521 threonine Nutrition 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 1
- MEYZYGMYMLNUHJ-UHFFFAOYSA-N tunicamycin Natural products CC(C)CCCCCCCCCC=CC(=O)NC1C(O)C(O)C(CC(O)C2OC(C(O)C2O)N3C=CC(=O)NC3=O)OC1OC4OC(CO)C(O)C(O)C4NC(=O)C MEYZYGMYMLNUHJ-UHFFFAOYSA-N 0.000 description 1
- 238000013060 ultrafiltration and diafiltration Methods 0.000 description 1
- 241000959836 unidentified Thermus OPB19 Species 0.000 description 1
- 241000959840 unidentified Thermus OPB31 Species 0.000 description 1
- 241000959843 unidentified Thermus OPB32 Species 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 235000014393 valine Nutrition 0.000 description 1
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 229920001221 xylan Polymers 0.000 description 1
- 150000004823 xylans Chemical class 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Images
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
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/975—Kit
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Enzymes And Modification Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
一態様では、本発明はTeg DNA Pol Iタンパク質を提供する。本発明のTegDNAポリメラーゼは、PCRにおける使用のための適当な、および一部の場合にはよりよい酵素を提供する。ここで示される通り、TegDNAポリメラーゼは他の一般的に用いられるDNAポリメラーゼI酵素に対して、より速い伸長速度、およびより高い忠実度を含む、顕著な長所を提供する。より高い忠実度の酵素は、正しい産物を増幅するのに、および、誤診および/または発現エラーを結果として生じうる、変異の導入を回避するのに必須である。さらに、より速い伸長速度は、PCR熱プロトコルに必要な時間を低減し、そのようにして実験効率を高めおよび一般費用を低減するのに決定的に重要である。
Kcat−酵素1モル当たりの単位時間に産物へ変換された基質の最大モル数
Kcat/KM−酵素ヌクレオチド複合体を産物へ変換する効率
Km−Vmax1/2の場合の基質濃度
好ましい一実施形態では、本発明はPol Iタンパク質変異体を提供する。これらの変異体は、3分類のうち一つ以上に該当する:置換、挿入および欠失変異体。これらの変異体は通常、Pol Iタンパク質をコードするDNA中のヌクレオチドの位置特異的変異誘発によってカセットまたはPCR変異誘発を用いて、または、変異体をコードするDNAを作製するための本分野でよく知られた他の方法によって、およびその後、上記で概説された通り、DNAを組み換え細胞培養で発現することによって、調製される。しかし、変異体タンパク質断片はまた、確立された方法を用いてin vitro合成によって調製されうる。アミノ酸配列変異体は、それらを天然に存在する対立遺伝子または種間Pol Iタンパク質と区別する性質である、変異の規定の性質によって特徴づけられる。一実施形態では、変異体は、天然に存在するアナログと同一の質的生物活性を示す。好ましい一実施形態では、改変された特徴を有する変異体が、下記により完全に概説される通り、提供される。
本発明の一部の実施形態では、TegDNAポリメラーゼIのドメインを、「融合タンパク質」を作製するのに使用できる。ここでは、「融合タンパク質」の語は、外来タンパク質断片と結合した目的タンパク質を含むキメラタンパク質をいう。融合パートナーは、宿主細胞で発現される通りの組み換えキメラタンパク質の溶解性を高めることができ、宿主細胞または培養上清からの組み換え融合タンパク質の精製を可能にする親和性タグを提供でき、または両方である。融合パートナーは、塩基誤取り込みを訂正する3'−5'−校正エキソヌクレアーゼ活性またはDNAテンプレートとの結合強度および親和性を改善するための追加のDNA結合部位といった、親ポリメラーゼには無い新規の望ましい機能を導入しうる。前者の場合のそのような融合パートナーの適した例は、古細菌校正DNAポリメラーゼの内部3'−5'−エキソヌクレアーゼドメインである。後者の場合の融合パートナーの例は、Ssod7のような好熱性古細菌由来の小熱安定性ヒストン様タンパク質、またはメタノコッカス・ジャナシイ(Methanococcus janaschii)由来DNAトポイソメラーゼの複数DNA結合ドメインである。必要に応じて、融合タンパク質は目的タンパク質から、本分野で公知であるさまざまな酵素的または化学的手段によって除去されうる。
一態様では、本発明は、核酸分子をTegDNAポリメラーゼIを含む複製反応混合物中で複製反応に供することを含む、核酸分子を複製する方法を提供する。
一態様では、本発明は、ここで開示されるTegDNAポリメラーゼIを含む増幅反応混合物中で核酸分子を増幅反応に供することを含む、核酸分子を増幅するための方法を提供する。好ましくは、増幅反応は、ここで記載される増幅反応チューブ中で実施される。
一態様では、本発明は、TegDNAポリメラーゼを含む配列決定反応混合物中での配列決定反応に核酸を供することを含む、核酸、好ましくはDNAを配列決定するための方法を提供する。
一態様では、本発明は、ここで開示される通りのTegDNAポリメラーゼIを利用する核酸複製のためのキットを提供する。本発明に記載のキットは、少なくとも、ここで開示される通りの本発明に係るDNAポリメラーゼIを含む。
すなわち:
5’−TCG AAT TCI CCY AAY TGR CCN T−3’(配列番号35)
Y = ピリミジン = C / T (縮重度= 2X)
R = プリン = A / G (縮重度 = 2X)
I = イノシン = C / G / A / T
N = ヌクレオチド = C / G / A / T (縮重度 = 4X)
本発明は、本発明のポリヌクレオチド分子を含むベクター、およびそのようなベクターで形質転換された宿主細胞を提供する。本発明のポリヌクレオチドのいずれも、一般的に選択可能なマーカーおよび複製開始点を含むベクターに含まれうる。ベクターは、細菌またはウイルス分子に由来するもののような、適当な転写および/または翻訳調節配列をさらに含む。そのような調節配列の例は、転写プロモーター、オペレーター、またはエンハンサー、mRNAリボソーム結合部位、および適当な配列を含む。プロモーターヌクレオチド配列がコード配列の転写を指示する場合、プロモーターヌクレオチド配列はコードDNA配列と調節可能に結合している。
本発明の新規ポリペプチド、およびそのセグメントは、ポリクローナルおよびモノクローナル抗体を生じるために使用されうる。抗体の設計および産生のための方法は本分野で知られており、たとえば、『抗体:実験の手引き』(Antibodies: A Laboratory Manual),ハーロー(Harlow)およびランド(Land)(編),1988コールド・スプリング・ハーバー・ラボラトリー・プレス社(Cold Spring Harbor Laboratory Press),ニューヨーク州コールド・スプリング・ハーバー(Cold Spring Harbor ,New York);『モノクローナル 抗体,ハイブリドーマ:生物分析の新次元』(Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analysis),ケネット(Kennet)他 (編),1980プレナム・プレス社(Plenum Press),ニューヨーク(New York)を参照。
計101検体の天然細菌分離体が、沿岸部(Snaefellsnes、OxarfjordurおよびReykjanes)、および低地(Hveragerdi)および高地領域(Hveravellir、HrafntinnuskerおよびHagongur)の両方を含む、アイスランドの8箇所の別々の地熱領域の水および堆積物から単離された。これらの場所は、植生および全体的な物理化学性が多様で、広い範囲のpHおよび温度、異なる水分活性および化学組成にわたった。採取から3時間後、試料をR2Aおよび160培地上に塗沫し、および72℃および78℃にて1〜2日インキュベートした(スキルニスドティル(Skirnisdottir)他2000c)(リーズナー(Reasoner)およびゲルドライヒ(Geldreich)1985)。淡黄色のコロニーを取り、および160培地上での反復した塗沫によって精製した。増殖および特性検査は液体160培地または160寒天プレート上で72℃にて実施した。160およびR2A培地上で72および78℃にて1日インキュベートした後、直径2〜3mmの淡黄色のコロニーが出現した。結果として生じたコロニーを160培地上でさらに精製した。
ファミリーI DNAポリメラーゼ、別名Pol Aは、ブレイズワイト(Braithwaite)およびイトー(Ito)(ブレイズワイト他,『DNAポリメラーゼの収集、アライメント、および系統分類的関係』(Compilation,Alignment,and Phylogentic Relationships of DNA Polymerases.) Nucleic Acids Res.21 (1993) 787−802.参照により本開示に含まれる)によって示唆された通り、これらのポリメラーゼの活性部位を形成する、保存された配列モチーフを含む。これらのモチーフは、ブロックA,BおよびCとしばしば呼ばれる、高度に保存された配列ブロックを含む (ジョイス(Joyce)他,『DNA ポリメラーゼにおける機能および構造関係』(Function and Structure Relationships in DNA Polymerases).Annu.Rev.Biochem.63 (1994) 777−822.,参照により本開示に含まれる)。サーマス(Thermus)種由来のいくつかのファミリーA DNAポリメラーゼのアライメントを、保存されたブロックAおよびCを用いて実施した。これらのモチーフ内の不変アミノ酸残基は、既知のサーマス(Thermus)ポリメラーゼIコード配列:A−forw:5’−GCCGCCGACTACTCCCARATHGARHT−3’(配列番号:36)およびC−rev:3’−CANGTRCTRCTCTACCACAAGCTCCCG−5’(配列番号:37)からの縮重プライマーを推論することを可能にした。
T.エガートソニイ(T.eggertssonii)#248株に由来する600bp polA遺伝子断片のヌクレオチド配列を用いて、ジーンマイニング(GENEMINING)(商標)と呼ばれるPCRを基礎とした遺伝子ウォーキング法を使用して、TegDNAポリメラーゼIの完全なコード配列をクローニングした。この方法は、既知の配列の3’末端の下流および既知の配列の5'末端の上流の未知の配列をそれぞれ増幅するための2組のプライマーを用いる。2組のプライマーのそれぞれは、既知のpolA配列から推論された遺伝子特異的プライマーおよび任意3’末端配列を有する反対プライマーを含む。第3のプライマーが両方の組に加えられ、それは任意プライマーの5’末端側半分の配列を含む。この配列はまた、PCR産物のクローニングを円滑化する2つの特異な制限部位を含む。3’末端が未知のpolA配列の増幅のためのプライマーセットでは、遺伝子特異的プライマーはフォワードプライマーであった。未知の5’末端のpolAの増幅のためのセットでは、遺伝子特異的プライマーはリバースプライマーであった。下記の3つの任意プライマーを使用した:
Arb1: 5'-GGCCACGCGTCGACTAGTACNNNNNNNNNNGATAT-3', (配列番号: 38)
Arb.2: 5'-GGCCACGCGTCGACTAGTACNNNNNNNNNNACGCC-3, (配列番号: 39)
Arb.3: 5'-GGCCACGCGTCGACTAGTAC-3'. (配列番号: 40)
完全長Teg、Tbr、TaqおよびTthDNAポリメラーゼのヌクレオチドおよびアミノ酸配列を、ソフトウェアパッケージ・ベクターNTI(インビトロジェン社(Invitrogen),カールスバード(Carslbad))のクラスタルW(Clustal W)アルゴリズムを用いてアライメントした。アライメントの結果を、DNA配列アライメントについて表6に、およびアミノ酸配列アライメントについて表7に要約する。完全なアミノ酸配列アライメントを図11aおよび11bに示し、および対応する系統樹を図12に示す。
TegDNAポリメラーゼの発現のためのPRI−pSO4ベクターは、TegDNAポリメラーゼを大腸菌(E.coli)において発現するために使用できる発現ベクタープラスミドの一種の具体例である。当業者は、大腸菌(E.coli)における、および酵母菌ピキア・パストリス(Pichia pastoris)、サッカロマイセス・セレビシエ(Saccharomyces cervisiae)またはクリベロマイセス・ラクチス(Klyveromyces lactis)といった他の宿主生物における、Teg DNAポリメラーゼの発現に適した非常にさまざまな発現ベクタープラスミドに詳しい。当業者にとって発現宿主の別の可能な選択は、不死化哺乳類細胞株および昆虫細胞株である。特定発現ベクターの選択は、宿主生物およびその遺伝的背景によって制限されおよび規定される。
TegDNAポリメラーゼを精製するための一般的精製手順の概要を図10に示す。該方法は4つの主な精製段階を用いる。最初に、粗細胞抽出物を75℃にてインキュベートして、非熱安定性大腸菌(E.coli)宿主タンパク質を変性および除去する。第2に、総核画分を熱処理細胞抽出物から、ポリエチレンイミン(PEI)を用いる沈澱によって除去する。第3に、清澄化した上清を、硫酸アンモニウム沈澱によって分画する。第4の段階は、ブチル−セファロースでの疎水性相互作用クロマトグラフィーを含み、および最後の段階は、ヘパリン−セファロース上でのリガンドアフィニティクロマトグラフィーを含む。
TaqDNAポリメラーゼの活性を測定するために開発された、非放射性の、PCRに基づく活性アッセイを用いた。アッセイは、ヒトゲノムDNAをテンプレートとして用いて増幅された、350bp PCR産物(ヒトベータアクチン遺伝子の断片)の終点定量に基づく。すべての反応物の量(各dNTP250μmol、テンプレートDNA50ng、プライマー40pmol、およびマグネシウムイオン2.0mM)は、DNAポリメラーゼを除いて、過剰に与えられ、そのため活性DNAポリメラーゼの量が産物収量の唯一の制限因子となる。各サイクルでのプライマー伸長およびアニーリングの時間は各8秒間の絶対最短値に設定された。サイクルの総数は30に設定され、ここで指数増幅期が産物飽和の段階に移行する。これらの条件下で、PCR産物の終点量は、反応50μl当たり0.2U〜0.8Uの範囲内でDNAポリメラーゼの入力量の線形関数である。市販TaqDNAポリメラーゼ(ロシュ・モレキュラー・ダイアグノスティカ(Roche Molecular Diagnostica);GMP−Taq)のロットが、製造元によって放射性プライマー伸長アッセイで5U/mlに調整されているが、本アッセイで酵素標準物質(enzyme calibrator)として用いられた。
DNAポリメラーゼの伸長速度は、プライミング(+)鎖が5’−3’方向へ伸長される時間間隔当たり塩基数(秒当たり塩基)によって測定される。プライマーの伸長を秒当たりごくわずかの塩基によって直接検出することは不可能なので、測定のための反応(重合)時間を延長するために長い1本鎖テンプレート分子が用いられる。この場合、7.2kb長1本鎖M13mp18DNAテンプレートを用いるM13ファージ複製アッセイを使用して、Teg−、Taq−およびTbrDNAポリメラーゼの伸長速度を比較して測定した。
熱安定性は熱安定性DNAポリメラーゼの最も重要な性能特性の1つである。好熱性微生物由来の起源は、PCRまたは熱サイクル配列決定反応における不可逆的熱不活性化に対するDNAポリメラーゼの十分な安定性に何ら保証を与えない。多数のDNAポリメラーゼが、新規の機能性またはより良好な性能特性を求めて、好熱性細菌から単離されているが、不十分な熱安定性がそれらのPCRでの有用性を限定している。そのような酵素の例は、ジオバチルス・ステロサーモフィルス(Geobacillus staerothermophilus)、サーモプラスマ・アシドフィラム(Thermoplasma acidophilum)およびアクイフェックス・スピーシーズ(Aquifex spec)種由来のI型DNAポリメラーゼである。そのリストに加えるべき別の一例がここでTbrDNAポリメラーゼによって示される。
DNAポリメラーゼの合成忠実度とは、プライミング鎖の3’末端での「間違った」ヌクレオチドの取り込みを識別する能力である。「間違った」ヌクレオチドとは、テンプレート鎖の対向塩基とのワトソン/クリック型水素結合を取れない塩基を持つヌクレオチドをいう。ポリメラーゼ活性部位における立体構造変化の熱力学的制限は、「間違った」塩基識別の基礎となる機構を提供する。立体構造的制限は、「非ワトソン−クリック」塩基対のDNAらせん歪みによって強制される。
Claims (6)
- プライミングされた1本鎖M13mp18DNAおよび60℃のインキュベーション温度を用いてDNA複製アッセイにおいて同一条件下で測定した場合、DNAポリメラーゼが>35塩基/秒であるインビトロ(in−vitro)プライマー伸長速度を有しおよびアミノ酸配列の配列番号:2または4を含むDNAポリメラーゼのプライマー伸長速度より速い、好熱性ポリメラーゼであって、
配列番号:6および44から成る群から選択されるアミノ酸配列からなるDNAポリメラーゼ。 - 請求項1に記載の精製されたDNAポリメラーゼのアミノ酸配列をコードする核酸。
- 配列番号:5または43の核酸配列を含むベクター。
- a)アミノ酸配列の配列番号:6、または44からなるDNAポリメラーゼをコードする単離された核酸に調節可能に結合しているプロモーター領域、
b)リボソーム結合部位、
c)選択可能な代謝マーカー遺伝子、
d)宿主細胞において機能的な複製開始点、および任意に
e)DNAポリメラーゼをコードする核酸配列転写物の翻訳を促進する3’非翻訳配列領域
を含む、請求項3に記載のベクター。 - a)請求項1に記載の好熱性DNAポリメラーゼI、任意に
b)反応緩衝液、および
c)任意にヌクレオチド
を含む核酸複製キット。 - DNA配列決定キットおよびDNA増幅キットの群から選択される、請求項5に記載の核酸複製キット。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP06021140A EP1914303A1 (en) | 2006-10-09 | 2006-10-09 | Thermus eggertssonii DNA polymerases |
EP06021140.6 | 2006-10-09 | ||
PCT/EP2007/060724 WO2008043765A1 (en) | 2006-10-09 | 2007-10-09 | Thermus eggertssonii dna polymerases |
Publications (3)
Publication Number | Publication Date |
---|---|
JP2010505434A JP2010505434A (ja) | 2010-02-25 |
JP2010505434A5 JP2010505434A5 (ja) | 2010-11-18 |
JP5394928B2 true JP5394928B2 (ja) | 2014-01-22 |
Family
ID=37564059
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2009531829A Expired - Fee Related JP5394928B2 (ja) | 2006-10-09 | 2007-10-09 | サーマス・エガートソニイ(thermuseggertssonii)dnaポリメラーゼ |
Country Status (6)
Country | Link |
---|---|
US (1) | US8183023B2 (ja) |
EP (2) | EP1914303A1 (ja) |
JP (1) | JP5394928B2 (ja) |
AT (1) | ATE477322T1 (ja) |
DE (1) | DE602007008449D1 (ja) |
WO (1) | WO2008043765A1 (ja) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2075331A1 (en) * | 2007-12-28 | 2009-07-01 | Qiagen GmbH | Modified enzymes and their uses |
JP5453613B2 (ja) * | 2008-09-30 | 2014-03-26 | 独立行政法人農業生物資源研究所 | 遺伝子クラスタリング装置およびプログラム |
JP6249400B2 (ja) * | 2011-07-12 | 2017-12-20 | 国立大学法人九州大学 | 新規dnaポリメラーゼ |
JP2013165669A (ja) * | 2012-02-15 | 2013-08-29 | Kyoto Univ | 変異型逆転写酵素 |
TWI468968B (zh) * | 2012-05-24 | 2015-01-11 | Nat Univ Chung Cheng | Based on the whole gene expression and biological response path and gene ontology reaction path grouping method |
Family Cites Families (34)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4179337A (en) * | 1973-07-20 | 1979-12-18 | Davis Frank F | Non-immunogenic polypeptides |
JPS6023084B2 (ja) * | 1979-07-11 | 1985-06-05 | 味の素株式会社 | 代用血液 |
US4469863A (en) * | 1980-11-12 | 1984-09-04 | Ts O Paul O P | Nonionic nucleic acid alkyl and aryl phosphonates and processes for manufacture and use thereof |
US4640835A (en) * | 1981-10-30 | 1987-02-03 | Nippon Chemiphar Company, Ltd. | Plasminogen activator derivatives |
DE3223104A1 (de) * | 1982-06-21 | 1983-12-22 | Hoechst Ag, 6230 Frankfurt | Photopolymerisierbares gemisch und damit hergestelltes photopolymerisierbares kopiermaterial |
US4496689A (en) * | 1983-12-27 | 1985-01-29 | Miles Laboratories, Inc. | Covalently attached complex of alpha-1-proteinase inhibitor with a water soluble polymer |
US5034506A (en) * | 1985-03-15 | 1991-07-23 | Anti-Gene Development Group | Uncharged morpholino-based polymers having achiral intersubunit linkages |
US5235033A (en) * | 1985-03-15 | 1993-08-10 | Anti-Gene Development Group | Alpha-morpholino ribonucleoside derivatives and polymers thereof |
US4683195A (en) * | 1986-01-30 | 1987-07-28 | Cetus Corporation | Process for amplifying, detecting, and/or-cloning nucleic acid sequences |
US4683202A (en) * | 1985-03-28 | 1987-07-28 | Cetus Corporation | Process for amplifying nucleic acid sequences |
EP0206448B1 (en) * | 1985-06-19 | 1990-11-14 | Ajinomoto Co., Inc. | Hemoglobin combined with a poly(alkylene oxide) |
US4791192A (en) * | 1986-06-26 | 1988-12-13 | Takeda Chemical Industries, Ltd. | Chemically modified protein with polyethyleneglycol |
US4962022A (en) * | 1986-09-22 | 1990-10-09 | Becton Dickinson And Company | Storage and use of liposomes |
CA1340807C (en) * | 1988-02-24 | 1999-11-02 | Lawrence T. Malek | Nucleic acid amplification process |
US5216141A (en) * | 1988-06-06 | 1993-06-01 | Benner Steven A | Oligonucleotide analogs containing sulfur linkages |
US5498523A (en) * | 1988-07-12 | 1996-03-12 | President And Fellows Of Harvard College | DNA sequencing with pyrophosphatase |
US4962020A (en) * | 1988-07-12 | 1990-10-09 | President And Fellows Of Harvard College | DNA sequencing |
US5168053A (en) | 1989-03-24 | 1992-12-01 | Yale University | Cleavage of targeted RNA by RNAase P |
US5386023A (en) * | 1990-07-27 | 1995-01-31 | Isis Pharmaceuticals | Backbone modified oligonucleotide analogs and preparation thereof through reductive coupling |
US5602240A (en) * | 1990-07-27 | 1997-02-11 | Ciba Geigy Ag. | Backbone modified oligonucleotide analogs |
US5210015A (en) * | 1990-08-06 | 1993-05-11 | Hoffman-La Roche Inc. | Homogeneous assay system using the nuclease activity of a nucleic acid polymerase |
US5455166A (en) * | 1991-01-31 | 1995-10-03 | Becton, Dickinson And Company | Strand displacement amplification |
US5644048A (en) * | 1992-01-10 | 1997-07-01 | Isis Pharmaceuticals, Inc. | Process for preparing phosphorothioate oligonucleotides |
US5436149A (en) * | 1993-02-19 | 1995-07-25 | Barnes; Wayne M. | Thermostable DNA polymerase with enhanced thermostability and enhanced length and efficiency of primer extension |
US5637684A (en) * | 1994-02-23 | 1997-06-10 | Isis Pharmaceuticals, Inc. | Phosphoramidate and phosphorothioamidate oligomeric compounds |
US5614365A (en) * | 1994-10-17 | 1997-03-25 | President & Fellow Of Harvard College | DNA polymerase having modified nucleotide binding site for DNA sequencing |
US6077664A (en) * | 1995-06-07 | 2000-06-20 | Promega Corporation | Thermophilic DNA polymerases from Thermotoga neapolitana |
US6001645A (en) * | 1995-06-07 | 1999-12-14 | Promega Corporation | Thermophilic DNA polymerases from thermotoga neapolitana |
US7056703B2 (en) | 1997-08-06 | 2006-06-06 | Diversa Corporation | Polypeptides having polymerase activity and methods of use thereof |
DE60027040T2 (de) * | 1999-10-29 | 2006-11-23 | Stratagene California, La Jolla | Zusammensetzungen und methoden zur verwendung von dna polymerasen |
AUPQ994600A0 (en) * | 2000-09-06 | 2000-09-28 | Agriculture Victoria Services Pty Ltd | Manipulation of plant senescene |
WO2003004632A2 (en) * | 2001-07-06 | 2003-01-16 | Amersham Biosciences Corp | Novel dna polymerases having amino acid substitutions and homologs thereof |
AU2002346498A1 (en) * | 2001-11-30 | 2003-06-17 | Applera Corporation | Thermus brockianus nucleic acid polymerases |
WO2006030455A1 (en) | 2004-09-17 | 2006-03-23 | Prokaria Ehf. | Dna polymerases having strand displacement activity |
-
2006
- 2006-10-09 EP EP06021140A patent/EP1914303A1/en not_active Withdrawn
-
2007
- 2007-10-09 JP JP2009531829A patent/JP5394928B2/ja not_active Expired - Fee Related
- 2007-10-09 WO PCT/EP2007/060724 patent/WO2008043765A1/en active Application Filing
- 2007-10-09 AT AT07821092T patent/ATE477322T1/de not_active IP Right Cessation
- 2007-10-09 EP EP07821092A patent/EP2074212B1/en not_active Not-in-force
- 2007-10-09 DE DE602007008449T patent/DE602007008449D1/de active Active
- 2007-10-09 US US12/442,600 patent/US8183023B2/en not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
---|---|
EP2074212A1 (en) | 2009-07-01 |
EP2074212B1 (en) | 2010-08-11 |
US20090317888A1 (en) | 2009-12-24 |
EP1914303A1 (en) | 2008-04-23 |
DE602007008449D1 (en) | 2010-09-23 |
US8183023B2 (en) | 2012-05-22 |
WO2008043765A1 (en) | 2008-04-17 |
ATE477322T1 (de) | 2010-08-15 |
JP2010505434A (ja) | 2010-02-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US5700672A (en) | Purified thermostable pyrococcus furiousus DNA ligase | |
US6489150B1 (en) | Purified thermostable Pyrococcus furiosus DNA polymerase I | |
US9896705B2 (en) | L-arabinose isomerase variants with improved conversion activity and method for production of D-tagatose using them | |
JP5394928B2 (ja) | サーマス・エガートソニイ(thermuseggertssonii)dnaポリメラーゼ | |
US5866395A (en) | Purified thermostable pyrococcus furiosus DNA polymerase I | |
JP2863738B2 (ja) | 熱安定性ピロホスファターゼをコードするdna | |
KR100777227B1 (ko) | 고호열성 dna 중합효소 및 이의 제조방법 | |
JP4308324B2 (ja) | テルモコッカス・ゴルゴナリウス由来の熱安定性核酸ポリメラーゼ | |
KR100777230B1 (ko) | 써모코커스 유래 돌연변이 dna 중합효소들 및 그의유전자들 | |
JP5617075B2 (ja) | 酵素 | |
KR101303990B1 (ko) | 써모코커스 파시피쿠스 균주 유래의 dna 중합효소 유전자 변이체들 및 이의 이용 | |
US20110020896A1 (en) | Mutant dna polymerases and their genes | |
US20240200040A1 (en) | Chimeric dna polymerase and application thereof | |
EP1075525B1 (en) | Dna polymerase from pyrobaculum islandicum | |
WO2007143436A2 (en) | Dna polymerase from spirochaeta thermophila | |
WO2008066350A1 (en) | A novel ditpase and genes encoding it | |
Dash | Functional Characterization of Pol-dUT Fusion Enzymes from Pyrococcus Furiosus | |
KR100774102B1 (ko) | 썰포포보코커스 질리지 유래의 내열성 dna 연결효소 | |
KR101142950B1 (ko) | 써모코커스 마리너스 균주 유래의 내열성 dna 중합효소 및 이의 이용 | |
KR101296882B1 (ko) | 써모코커스 와이오타푸엔시스 균주 유래의 dna 중합효소 변이체들 및 이의 이용 | |
Slobodkina et al. | Isolation and characterization of thermostable DNA polymerase of the hyperthermophilic archaeum Thermococcus litoralis Sh1AM | |
KR20100107873A (ko) | 써모코커스 씰러 균주 유래의 내열성 dna 중합효소 및 이의 이용 | |
KR20110103750A (ko) | 써모코커스 래디오톨러런스 균주 유래의 내열성 dna 중합효소 및 이의 이용 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20100929 |
|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20100929 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20121002 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20121227 |
|
A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20130402 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20130801 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20130911 |
|
A911 | Transfer to examiner for re-examination before appeal (zenchi) |
Free format text: JAPANESE INTERMEDIATE CODE: A911 Effective date: 20130917 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20131008 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20131017 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 5394928 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
LAPS | Cancellation because of no payment of annual fees |