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Transcript of JAS 分析試験ハンドブック - famic.go.jp · PDF filejas 分析試験ハンドブック...

  • JAS

    3

  • IP

    PCR

  • 13 4 1

    PCR

    13 5 25 . PCR

    14 6 20

    (

    )

    PCR

    PCR (

    )

    PCR DNA

    24 9 24

    SSIIb-3)

  • PCR

    PrimerProbe Mix (SSIIb-3)

    DNA ColE1/TE GM (RRS) GM

    NTC ColE1/TE

    3.

  • i) 1ii) 1iii) 2 2 2 DNA 2 DNeasy Plant Maxi kit DNA 2 2 2 2 2 3 3 DNeasy Plant Maxi kit DNA 3 DNeasy Plant Maxi kit DNA 4

    DNA DNA 4 DNA 4 5 5

    QIAGEN Genomic-tip 20/G DNA 5 5 5 5 6 6 QIAGEN Genomic-tip 20/G DNA 7 DNA DNA 7 DNA 7 7 7

    CTAB DNA 7 7 7 8 8 8 8

  • DNA DNA 9 DNA 9 9

    PCR 9 9 10 10 10 PCR 10 10

    11 PCR 11 12

    12 PCR 12 13 13 13

    PCR 14 14 14

    15

    . 19. 19. 19. 20. 20. 20 21 22

    i) 23ii) 23 23 24 24 24

  • 24 24 25 25 25 26 26 26 26 26 26 27

    27 27 27 28 28 28 28 28 28 28 29 29 29

    29 29

    29 29 30 30 30 30

    30 31

  • PCR 33 (GM) 33 34 34 35 37

    39 39 39 39 DNA 39 39

    40 DNA 40 40

    41 41 41 DNA 41

    42 42 44

    45 46 46 46 46

    1. Th.Line 49 2. 50 3. 51

    53 53 54 0.5 mol/L EDTA (pH8.0) 54 1 mol/L Tris-HCl 54 0.5 mol/L Tris-HCl (pH 8.0) 55

  • 0.1 mol/L Tris-HCl (pH 8.0) 55 TE 55 CTAB 56 CIA 56 PCI 5610 5711 TAE 5812 TBE 5813 DNA 5914 5915 K 60

    61 61 61. 61. DNA 62. PCR 62. 62

    63. 63. 63. 63. 63. 63. 64

  • - 1 -

    i)

    JAS

    IP

    polymerase chain reaction, PCR

    Institute for Reference Materials and Measurements,IRMMAmerican Oil Chemists' Society, AOCScertified reference materials, CRMshouse referencematerials, HRMs

    deoxyribonucleic acid, DNA

    PCR

    ii) PCR DNA DNA PCR

    DNA deoxyribonuclease, DNase

    0.0056 mS/m25 C 121 C15

    1.5 mL 0.2 mL

    DNA

  • - 2 -

    Tris-EDTATE DNase

    iii)

    DNA

    cetyl-trimethyl ammonium bromide, CTAB DNAPCR DNA

    QIAGEN DNeasy Plant Maxi kit# 68163 QIAGENGenomic-tip 20/G# 10223DNeasy Plant Maxi kit Genomic-tip 20/G

    DNA

    DNeasy Plant Maxi kit DNA

    RNA ribonuclease, RNaseDNA

    Kuribara, Hideo; Shindo, Yoichiro; Matsuoka, Takeshi; Takubo, Ken; Futo, Satoshi; Aoki, Nobutaro; Hirao,Takashi; Akiyama, Hiroshi; Goda, Yukihiro; Toyoda, Masatake; Hino, Akihiro. Novel Reference Molecules forQuantitation of Genetically Modified Maize and Soybean. J. AOAC Int. 2002, vol. 85, p. 1077-1089. DNA

  • - 3 -

    50 mL 3,000g2 mL0.5-10 L10-100 L100-1,000 L1,000-5,000 L

    QIAGEN DNeasy Plant Maxi kit

    DNeasy Plant Maxi kit DNA 50 mL10-100 L 20 L

    RNasekit 1,000-5,000 L 10 mL Buffer AP1kit65 C

    65 C 115 10

    3,000g 101,000-5,000 L

    7 mL 15 mL 50 mL1,000-5,000 L 2.5 mL Buffer P3AP2

    bufferkit 10 15

    3,000g 351,000-5,000 L

    8 mLQIA shredder spin column lilac3,000g 51,000-5,000 L

    7.5 mL 50 mL10 101,000-5,000 L

    6.8 mL 50 mL111,000-5,000 L 10.2 mL Buffer AW1AP3/Et-OH

    bufferkit 10 DNeasy spin columncolorless

    123,000g 15131,000-5,000 L 12 mL Buffer AW2 AW

    bufferkit 3,000g 15

    14 50 mL100-1,000 L 1 mL 65 C

    155 3,000g 10 16100-1,000 L2 mL

    100-1,000 L

  • - 4 -

    17 1051812,000g 4 C15100-1,000 L

    19100-1,000 L 500 L 70 %

    2012,000g 4 C3100-1,000 L 10-100 L

    2110-100 L 100 L TE (pH 8.0)

    2212-24

    23 DNA 24 12,000g 4 C3 DNA-20 C

    DNeasy Plant Maxi kit DNA 50 mL0.5-10 L 10 L

    RNase (kit) 1,000-5,000 L 5 mL Buffer AP165C

    65 C 115 10

    1,000-5,000 L1.8 mL Buffer P3AP2 buffer 10 15

    3,000g 151,000-5,000 L

    4.2 mLQIA shredder spin column lilac3,000g 51,000-5,000 L

    4 mL 50 mL 101,000-5,000 L

    3.4 mL 50 mL1,000-5,000 L 5.1 mL Buffer AW1AP3/Et-OH

    buffer 10 DNeasy spin columncolorless

    103,000g 51121 DNeasy Plant Maxi kit DNA

    1323

    DNA DNA DNA0.5-10 L 5 LTE

    50 L 200 300 nm230260 280 nm1 O.D. 260 nm 50 ng/L DNA DNA

  • - 5 -

    PCR10 ng/LDNA

    DNAO.D.260 nm/ O.D.280 nm 1.7 2.0

    PCR DNA

    DNeasy Plant Maxi kit DNA 21DNA TE 100 LTE 50 LPCR DNA

    DNA DNA DNeasy Plant Maxi kit DNA

    21DNA TE 20 LPCR DNA DNA PCR DNAPCR DNA DNA

    DNA O.D.260 nm/O.D.230 nmPCR DNA DNA PCR

    DNA230 nm260 nm280 nm

    Sambrook, J., Russel, D. W. "Molecular cloning : a laboratory manual, 3rd Ed. (volume 3)", ColdSpring Harbor, N.Y., Cold Spring Harbor Laboratory Press, 2001, A8.20. (ISBN 0-87969-577-3 (pbk),ISBN 0-87969-576-5 (cloth))

    DNA

    QIAGEN Genomic-tip 20/G DNA

    RNase DNA

    DNA DNA

  • - 6 -

    50 mL 3,000g2 mL0.5-10 L10-100 L100-1,000 L1,000-5,000 L

    QIAGEN QIAGEN Genomic-tip 20/G# 10223

    RNase AQIAGEN RNase A# 19101Proteinase KQIAGEN QIAGEN Proteinase K# 19131 19133G2QBTQC QF

    QIAGEN Genomic-tip 20/G DNA 50 mL 1,000-5,000 L 7.5

    mL G21,000-5,000L 7.5 mL G2

    100-1,000 L 200 L QIAGEN Proteinase K 10-100 L 20 L RNase A

    50 C 115 10

    3,000g 4 C 1515 mL 50 mL1,000-5,000 L

    QIAGEN Genomic-tip 20/G 1 mL QBT100-1,000 L 1,000-5,000 L

    2 mL QIAGEN Genomic-tip 20/G

    tip100-1,000 L 1,000-5,000 L2 mL QC

    1011tip 1.5 mL100-1,000 L 750 L QF

    (50 C)DNA12tip 1.5mL100-1,000 L 750 L

    QF(50 C)DNA13 100-1,000 L

    10 5

  • - 7 -

    14()12,000g 4 C15200-1,000 L

    15100-1,000 L 1,000 L 70 % 10

    1612,000g 4 C3100-1,000 L 10-100 L

    1710-100 L 50 L TE (pH 8.0) 65 C 15

    1810-100 L DNA 65 C 15

    19(12-24) 20 DNA 24

    12,000g 4 C3 DNA-20 C

    2127 DNeasy Plant Maxi kit DNA1723

    DNA DNA

    DNAO.D.260 nm/ O.D.280 nm 1.7 2.0

    DNeasy Plant Maxi kit DNA21 QIAGEN Genomic-tip 20/G DNA 17

    Blood & Cell Culture DNA Mini Kit#13323Genomic DNA buffer set# 19060

    CTAB DNA

    RNase DNA CTAB

  • - 8 -

    Murray, M.G.; Thompson, W.F. Rapid isolation of high molecular weight plant DNA. Nucl. Acids Res. 1980,vol. 8, p. 4321-4325.

    2 mL0.5-10 L10-100 L100-1,000 L1,000-5,000 L

    CTAB 2 mL1.5mL60 C3014,000 rpm3 700 L

    --PCI PCI214,000 rpm15

    -CIA CIA214,000 rpm3

    30 12,000 rpm3

    70 % 800 L312,000 rpm3

    105 11DNA RNATE 100 lRNase A10 mg/mL2 LDNA 37 C 30400 L CTAB

    CIA500 l CIA12,000 rpm15

    30 12,000 rpm3

  • - 9 -

    105 11DNA

    100 LDNADNA-20 C 12 13

    DNA DNA

    DNAO.D.260 nm/ O.D.280 nm 1.8 2.0

    PCI

    PCI K20 mg/mL 20 L

    Centrifuge 5417REppendorf 16,000g

    PCI CTAB

    DNA

    13,000g 105,000 12,000 rpm

    11 12DNA TETE EDTA PCR

    PCR

    13

  • - 10 -

    PCR

    DNA DNA PCR PCR DNA PCR PCR PCR

    DNA

    PCRKuribara, Hideo; Shindo, Yoichiro; Matsuoka,Takeshi; Takubo, Ken; Futo, Satoshi; Aoki, Nobutaro; Hirao, Takashi; Akiyama, Hiroshi; Goda, Yukihiro; Toyoda,Masatake; Hino, Akihiro. Novel Reference Molecules for Quantitation of Genetically Modified Maize andSoybean. J. AOAC Int. 2002, vol. 85, p. 1077-1089 Kodama, Takashi; Kuribara, Hideo; Minegishi,Yasutaka; Futo, Satoshi; Watai, Masatoshi; Sawada, Chihiro; Watanabe, Takahiro; Akiyama, Hiroshi; Maitani,Tamio; Teshima, Reiko; Furui, Satoshi; Hino, Akihiro; Kitta, Kazumi. Evaluation of Modified PCR Quantitationof Genetically Modified Maize and Soybean Using Reference Molecules: Interlaboratory Study. J. AOAC Int.2009, vol. 92, p. 223-233. DNA

    RoundupReady Soy40-3-2 Bt11Event176T25MON810 GA21

    NewLeafBt6 SPBT02-05 NewLeaf PlusRBMT21-129, RBMT21-350 RBMT22-82

    Cauliflower mosaic virus 35S promoter

    PCR MJ Research PTC-200 DNA Engine, TaKaRa PCR Thermal Cycler

    MP, Applid Biosystems GeneAmp system 9700: MAX PCR

    0.5-10 L10-100 L100-1,000 L1,000-5,000 L

  • - 11 -

    II

    0.5-10 L10-100 L100-1,000 L1,000-5,000 L

    PCRDNAAmpliTaqTM GoldApplied BiosystemsdNTP2 mmol/L eachAmpliTaqTM Gold MgCl225 mmol/LAmpliTaq

    TM Gold PCR10 PCR buffer II

    (Le1)# 313-05501# S1-1M

    RRS specific# 310-05511# S2-1M

    SSIIb# 315-05441# 312-06051# M1-1M

    CaMV 35S promoter # 317-05521# C1-1MGA21 specific # 312-05451# M2-1M

    SSIIb# 315-05441# 312-06051# M1-1M

    GA21 specific # 312-05451# M2-1MBt11 specific # 319-05461# M3-1MEvent176 specific # 316-05471# M4-1MT25 specific # 313-05481# M5-1MMON810 specific # 310-05491# M6-1M

    Pss# 316-05231#G5-1

    NewLeaf specificNewLeaf Plus specific # 312-05191# G1-1

  • - 12 -

    GM RRS# 311-04941# PS-1

    GM # 314-04811# PM-1

    GM NewLeaf Plus#311-05301# PP-1

    Tris--EDTATBE Tris--EDTATAE

    DNAPCR100 bp

    PCR

    121 C15

    PCRPCR DNA DNA PCR

    PCR PCR DNA

    DNA PCR

    PCR PCR PCR 0.2 mL

    DNA 22.5 L

  • - 13 -

    DNA 2.5 L DNA

    PCRPCR PCR

    II

    2 3 %TBE 10 11 12100 mL 50 g 1330 14

    DNA 15TBE 10 16

    PCR DNA 5 L 1 L 17 DNA 18

    100 V 19bromophenol blue, BPB 1/2 20

    21

    2223CCDcharge coupled deviceDNA

  • - 14 -

    2 3 %TBE 10 11 12 1330 14

    DNA 15TBE 10 16

    PCR DNA 5 L 1 L 17 DNA 18

    100 V 19 BPB 1/2 20

    21

    100 mL 50 g 30

    22 23CCD DNA

    PCR

    DNA PCR

    DNeasy Plant Maxi kit DNA PCR

    SSIIb SSIIb-1 SSIIb-3 SSIIb-3 SSIIb-1 DNA

  • - 15 -

    NewLeaf NewLeaf CaMV 35Spromoter GM RRSGM cauliflower mosaic virus, CMVPCR

    PCR

    PCRPC