2-1 DNA 1 DNA(plasmid DNA) pet21ahmyh hmyh (6A C) BL-21 Gold strain, 2003 2-1 pet21ahmyh Qiagen mini-preparation kit DNA DNA DNA( hmyh ) DNA( hmyh ) DNA DNA 2-2 DNA DNA AS-PCR 2 DNA DNA genomic DNA 30 genomic DNA -20 2-1 13
30 (A C) 2-1 pet21ahmyh hmyh (A) hmyh DNA(B) DNA 1 1 (6A C) (A) 2-2 (A) hmyh (B) DNA (6A C) (B) 14
2-1 30 Characteristics Number ( % ) 14 ( 46.7 % ) 16 ( 53.3 % ) ( ) Mean : 67.7, Range( 32 ~ 86 ) <50 3 ( 10.0 % ) 50-60 5 ( 16.7 % ) 60-70 8 ( 26.7 % ) 70-80 7 ( 23.3 % ) >80 7 ( 23.3 % ) 2-2 hmyh 2-2 AS-PCR G382D Y165C V232F R227W 3 2-3 2-4 Y165C 3 3-3.2 3 15
表 2-2 AS-PCR 之引子序列 Primer name Primer sequence 5 à3 THU-MYH-5 5'-CCAACACTGGACAGTGCCACCT-3' WT Reverse THU-MYH-6 5'-ACAGTCCTGCCAGCAGAC-3' MT Reverse THU-MYH-7 5'-ACAGTCCTGCCAGCAGAT-3' THU-MYH-1 5'-CTACTATACCGGATGGATGC-3' WT Reverse THU-MYH-2 5'-CGCCGGCCACGAGAATAGT-3' MT Reverse THU-MYH-3 5'-CGCCGGCCACGAGAATAGC-3' THU-MYH-1 5'-CTACTATACCGGATGGATGC-3' WT Reverse THU-MYH-12 5'-TCAGCACCAATGGCTCGGAC-3' MT Reverse THU-MYH-13 5'-TCAGCACCAATGGCTCGGAA-3' THU-MYH-1 5'-CTACTATACCGGATGGATGC-3' WT Reverse THU-MYH-10 5'-CTCGGACACGGCACAGCACCCG-3' MT Reverse THU-MYH-11 5'-CTCGGACACGGCACAGCACCCA-3' WT Primer 1 5'-GTCGCGGATCCATGACA-3' MT Primer 2 5'-GTCGCGGATCCATGACC-3 WT reverse THU-MYH-2 5'-CGCCGGCCACGAGAATAGT-3' Mutation G382D Y165C V232F R227W 質體 DNA 點突變處 WT 表示為 wild type specific 野生型特異性引子 MT 表示 mutant specific 突變型特異性引子 表 2-3 定序引子序列 Primer name Primer sequence 5 à3 THU-MYH-5 5'-CCAACACTGGACAGTGCCACCT-3' Reverse THU-MYH-8 5'-CCTTGCTGGGCTACTATTCT-3' THU-MYH-53 5'-AAGTGGCCTACACTGCAGGA-3' Reverse THU-MYH-54 5'-CGCTGTGGGGTACACACTGT-3' THU-MYH-53 5'-AAGTGGCCTACACTGCAGGA-3' Reverse THU-MYH-54 5'-CGCTGTGGGGTACACACTGT-3' Mutation G382D Y165C V232F 16
2-4 Y165C Mutation Primer name Primer sequence 5 3 Y165C THU-MYH-1 5'-CTACTATACCGGATGGATGC-3' WT Reverse THU-MYH-2 5'-CGCCGGCCACGAGAATAGT-3' MT Reverse THU-MYH-3 5'-CGCCGGCCACGAGAATAGC-3' Reverse THU-MYH-2M 5'-CGCCGGCCACGAGAATACT-3' Reverse THU-MYH-3M 5'-CGCCGGCCACGAGAATACC-3' Reverse THU-MYH-2P 5'-CGCCGGCCACGAGAATAGT(P)-3' Reverse THU-MYH-3P 5'-CGCCGGCCACGAGAATAGC(P)-3' Reverse THU-MYH-DJ1 5'-CGCCGGCCACGAGAATAGC(aa)-3 ' WT wild type specific MT mutant specific (P) (aa) AminoC6 2-3 2-5 2-5 Agar Agarose Ampicillin Ethidium bromide Ethyl alcohol USB (Cleveland, OH,USA) USB (Cleveland, OH,USA) Sigma (St. Louis, MO, USA) USB (Cleveland, OH,USA) Showa (Tokyo, Japan) US10906 32803 A-0166 US32813 0502-3160 17
Glycerol Hydrochloric acid QIAprep spin Miniprep Kit Sodium chloride TAE buffer 50X Tryptone Yeast extract NIHON SHIYAKU (Osaka, Japan) Merck (Darmstadt, Germany) Qiagen (Hilden, Germany) Merck (Darmstadt, Germany) Merck (Darmstadt, Germany) USB (Cleveland, OH,USA) USB (Cleveland, OH,USA) 170292 1.00317.2000 27106 1.06404.1000 444125D US12855 US23547 18
2-4 2-6 2-3 DNA Marker 2-6 100 bp ET marker Blossom GPM04 (Taipei,Taiwan) 100 Base Pair DNA ladder MDBio WSDL100 datp dttp dctp dgtp Taq DNA polymerase Pfx Vent (Taipei, Taiwan) MDBio (Taipei, Taiwan) Stratagene (La Jolla, USA) N0446S N04466S N04466S N04466S 101-9012-90-2 600380 M0254S 19
Restriction enzyme BamH I Restriction enzyme Xho I Restriction enzyme Bgl II R0136S R0146S R0144S (A) 2-3 DNA Marker (A) (B) 100 bp ET marker (Blossom) 100 Base Pair DNA ladder(mdbio) (B) 20
2-5 2-7 2-7 (micro-pipette) Eppendorf (Hamburg, Germany) Research 1000/200/100/20/2.5 Spectronic instruments Genesys2 (UV/visible Spectrophotometer) (Autoclaver) (micro-electronic Balance Meter) ( 4 C) (refrigerator) ( -20 C) (Low temperature refrigerator) ( -80 C) (Ultra-low temperature Freezer) (power supply) DNA (DNA electrophoresis cell) (New York, USA) Trident (California, USA) OHAUS (New Jersey, USA) TECO (Taipei, Taiwan ) Fisher&Paykel (Queensland, Australia) Thermo (Waltham, USA) Bio-Rad (Hercules, USA) Amersham Bioscience (Buckinghamshire, UK) EA-635 AR1530 RE7222 Standard H160 Forma-86 ULT Freezer Power Pac Basic Hoefer HE33 DNA/ Kodak DC290 (DNA/protein Imaging system) (Taipei, Taiwan) 21
DNA/ Kodak 1D Image Analysis (DNA/protein Imaging analysis (Taipei, Taiwan) system) Applied Biosystems Gene Amp PCR (PCR instrument) (centrifuge) (DIamond Ultrapure Water system) 2/3D (2/3D Waver) (Vortex-Mixer) (Foster, USA) Eppendorf (Hamburg, Germany) Barnstead (Dubuque, Iowa, USA) MAJOR SCIENCE (Taipei, Taiwan ) Scientific Industries (New York, USA) System9700 5415D NANO pure Dlamond TM MW-23 VORTEX-2 GENIE 22