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  3. Luận văn: BƯỚC ĐẦU XÂY DỰNG QUY TRÌNH ĐỊNH LƯỢNG CÁC SẢN PHẨM BIẾN ĐỔI GEN BẰNG PHƯƠNG PHÁP REAL-TIME PCR (part 10)

Luận văn: BƯỚC ĐẦU XÂY DỰNG QUY TRÌNH ĐỊNH LƯỢNG CÁC SẢN PHẨM BIẾN ĐỔI GEN BẰNG PHƯƠNG PHÁP REAL-TIME PCR (part 10)

Vào thế kỷ 20, sinh học đã có những bước tiến vượt bậc trong việc nghiên cứu sinh học phân tử ở mức độ DNA và protein. Các tiến bộ này đã được ứng dụng vào thực tiễn và phát triển thành một công nghệ mới, đó là công nghệ gen, bao gồm: các kỹ thuật tái tổ hợp DNA và chuyển nạp gen. 104 Gen tham chiếu: PCR Standard Curve Report with PCR Amp Cycle Graph PCR Base Line Subtracted Data Current Date: 19-Aug-05 12:05 PM Data generated on: 22-Jul-05 at 06:15 PM. Optical data file name: Data 22-Jul-05 181

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  1. 104 Gen tham chiếu: PCR Standard Curve Report with PCR Amp Cycle Graph PCR Base Line Subtracted Data Current Date: 19-Aug-05 12:05 PM Data generated on: 22-Jul-05 at 06:15 PM. Optical data file name: Data 22-Jul-05 1815-Truong-35S-dot 1-sua2.odm Plate Setup file used: GMO-35S.psm Protocol file used: GMO-35S.tmo Sample volume: 25.00 ul Hot Start? No W ell factor collection: Experimental Plate Comments Protocol Cycle 1: ( 1X) 95.0ºC Step 1: for 10:00 Cycle 2: ( 45X) 95.0ºC Step 1: for 00:15 60.0ºC Step 2: for 01:00 Data collection and real-time analysis enabled. Cycle 3: ( 1X) 4.0ºC Step 1: HOLD PCR Amp/Cycle Graph for FAM -490 Standard Curve Graph for FAM -490 104
  2. 105 Data Analysis Parameters Calculated threshold has been replaced by the user selected threshold 67.5. User selected baseline cycles are 2 to 20. Data analysis window is set at 95.00% of a cycle, centered at end of the cycle. Weighted Mean digital filtering has been applied. Global filtering is off. Standard Curve Spreadsheet Data for FAM -490 Units: copy number Type Identifier Rep Ct Log SQ SQ SQ Ct Ct Set SQ Mean SD Mean SD Point B02 Standard Stan Corn-Ref 1 28.29 4.612 4.10E+04 4.10E+04 N/A 28.29 N/A B04 Standard Stan Corn-Ref 2 30.26 3.709 5.12E+03 5.12E+03 N/A 30.26 N/A B06 Standard Stan Corn-Ref 3 32.97 2.806 6.40E+02 6.40E+02 N/A 32.97 N/A B08 Standard Stan Corn-Ref 4 35.43 1.903 8.00E+01 8.00E+01 N/A 35.43 N/A B11 Unknown Bt 9 28.93 4.308 2.03E+04 2.03E+04 N/A 28.93 N/A Đợt 3: Định lƣợng bắp chuyển gen Bt 176 1% (lần 2) Promoter 35S: PCR Standard Curve Report with PCR Amp Cycle Graph PCR Base Line Subtracted Curve Fit Data Current Date: 19-Aug-05 12:17 PM Data generated on: 16-Aug-05 at 04:58 PM. Optical data file name: Data 16-Aug-05 1658 taqman dot 3-35S -Bt176.odm Plate Setup file used: 35S TAQMAN DOT 3.psm Protocol file used: GMO-35S.tmo Sample volume: 25.00 ul Hot Start? No W ell factor collection: Experimental Plate Comments Protocol Cycle 1: ( 1X) 95.0ºC Step 1: for 10:00 Cycle 2: ( 45X) 95.0ºC Step 1: for 00:15 60.0ºC Step 2: for 01:00 Data collection and real-time analysis enabled. Cycle 3: ( 1X) 4.0ºC Step 1: HOLD PCR Amp/Cycle Graph for FAM -490 105
  3. 106 Standard Curve Graph for FAM -490 Data Analysis Parameters Calculated threshold has been replaced by the user selected threshold 21.8. Per-well baseline cycles have been determined automatically. Data analysis window is set at 95.00% of a cycle, centered at end of the cycle. Weighted Mean digital filtering has been applied. Global filtering is off. Standard Curve Spreadsheet Data for FAM -490 Units: copy number Type Identifier Rep Ct Log SQ SQ SQ Ct Ct Set SQ Mean SD Mean SD Point E02 Standard ST 35S 5 23.85 3.709 5.12E+03 5.12E+03 N/A 23.85 N/A E03 Standard ST 35S 6 26.55 2.806 6.40E+02 6.40E+02 N/A 26.55 N/A E04 Standard ST 35S 7 29.13 1.903 8.00E+01 8.00E+01 N/A 29.13 N/A E05 Standard ST 35S 8 31.67 1.000 1.00E+01 1.00E+01 N/A 31.67 N/A F02 Unknown Bt 19 28.56 2.092 1.24E+02 1.24E+02 N/A 28.56 N/A Gen tham chiếu: PCR Standard Curve Report with PCR Amp Cycle Graph PCR Base Line Subtracted Data Current Date: 19-Aug-05 12:22 PM Data generated on: 16-Aug-05 at 04:58 PM. Optical data file name: Data 16-Aug-05 1658 taqman dot 3-DC Bt 176.odm Plate Setup file used: 35S TAQMAN DOT 3.psm Protocol file used: GMO-35S.tmo Sample volume: 25.00 ul 106
  4. 107 Hot Start? No W ell factor collection: Experimental Plate Comments Protocol Cycle 1: ( 1X) 95.0ºC Step 1: for 10:00 Cycle 2: ( 45X) 95.0ºC Step 1: for 00:15 60.0ºC Step 2: for 01:00 Data collection and real-time analysis enabled. Cycle 3: ( 1X) 4.0ºC Step 1: HOLD PCR Amp/Cycle Graph for FAM -490 Standard Curve Graph for FAM -490 Data Analysis Parameters Calculated threshold has been replaced by the user selected threshold 21.2. User selected baseline cycles are 2 to 20. Data analysis window is set at 95.00% of a cycle, centered at end of the cycle. Weighted Mean digital filtering has been applied. Global filtering is off. Standard Curve Spreadsheet Data for FAM -490 Units: copy number Type Identifier Rep Ct Log SQ SQ SQ Ct Ct Set SQ Mean SD Mean SD Point B02 Standard ST CR 1 23.31 4.612 4.10E+04 4.10E+04 N/A 23.31 N/A B03 Standard ST CR 2 25.71 3.709 5.12E+03 5.12E+03 N/A 25.71 N/A B04 Standard ST CR 3 27.04 2.806 6.40E+02 6.40E+02 N/A 27.04 N/A 107
  5. 108 B05 Standard ST CR 4 30.64 1.903 8.00E+01 8.00E+01 N/A 30.64 N/A C02 Unknown Bt 3 24.51 4.097 1.25E+04 1.25E+04 N/A 24.51 N/A Đợt 4: Độ nhạy của phản ứng PCR Standard Curve Report with PCR Amp Cycle Graph PCR Base Line Subtracted Curve Fit Data Current Date: 30-Aug-05 06:46 AM Data generated on: 22-Jul-05 at 06:15 PM. Optical data file name: Data 22-Jul-05 1815-Truong-35S-dot 1-do nhay.odm Plate Setup file used: GMO-35S.psm Protocol file used: GMO-35S.tmo Sample volume: 25.00 ul Hot Start? No Well factor collection: Experimental Plate Comments Protocol Cycle 1: ( 1X) 95.0ºC Step 1: for 10:00 Cycle 2: ( 45X) 95.0ºC Step 1: for 00:15 60.0ºC Step 2: for 01:00 Data collection and real-time analysis enabled. Cycle 3: ( 1X) 4.0ºC Step 1: HOLD PCR Amp/Cycle Graph for FAM-490 Standard Curve Graph for FAM-490 108
  6. 109 Data Analysis Parameters Calculated threshold has been replaced by the user selected threshold 13.7. User selected baseline cycles are 2 to 24. Data analysis window is set at 95.00% of a cycle, centered at end of the cycle. Weighted Mean digital filtering has been applied. Global filtering is off. Standard Curve Spreadsheet Data for FAM-490 Units: copy number Type Identifier Rep Ct Log SQ SQ SQ Ct Ct Set SQ Mean SD Mean SD Point E02 Standard Stan Corn-Ref 5 25.73 3.709 5.12E+03 5.12E+03 N/A 25.73 N/A E04 Standard Stan Corn-Ref 6 28.57 2.806 6.40E+02 6.40E+02 N/A 28.57 N/A E06 Standard Stan Corn-Ref 7 31.65 1.903 8.00E+01 8.00E+01 N/A 31.65 N/A E08 Standard Stan Corn-Ref 8 33.12 1.000 1.00E+01 1.00E+01 N/A 33.12 N/A F08 Unknown Mau 1 copy 4 36.22 0.047 1.11E+00 1.25E+00 1.91E-01 36.09 1.87E-01 F10 Unknown Mau 1 copy 4 35.96 0.141 1.38E+00 1.25E+00 1.91E-01 36.09 1.87E-01 Đợt 5: Định lƣợng các mẫu thí nghiệm (lần 1) Promoter 35S: PCR Standard Curve Report with PCR Amp Cycle Graph PCR Base Line Subtracted Curve Fit Data Current Date: 11-Aug-05 03:43 PM Data generated on: 22-Jul-05 at 06:15 PM. Optical data file name: Data 22-Jul-05 1815-Truong-35S-dot 1-sua.odm Plate Setup file used: GMO-35S.psm Protocol file used: GMO-35S.tmo Sample volume: 25.00 ul Hot Start? No W ell factor collection: Experimental Plate Comments Protocol 109
  7. 110 Cycle 1: ( 1X) 95.0ºC Step 1: for 10:00 Cycle 2: ( 45X) 95.0ºC Step 1: for 00:15 60.0ºC Step 2: for 01:00 Data collection and real-time analysis enabled. Cycle 3: ( 1X) 4.0ºC Step 1: HOLD PCR Amp/Cycle Graph for FAM -490 Standard Curve Graph for FAM-490 Data Analysis Parameters Calculated threshold has been replaced by the user selected threshold 52.2. User selected baseline cycles are 2 to 24. Data analysis window is set at 95.00% of a cycle, centered at end of the cycle. Weighted Mean digital filtering has been applied. Global filtering is off. Standard Curve Spreadsheet Data for FAM -490 Units: copy number Type Identifier Rep Ct Log SQ SQ SQ Ct Ct Set SQ Mean SD Mean SD Point E02 Standard Stan Corn-Ref 5 27.43 3.709 5.12E+03 5.12E+03 N/A 27.43 N/A E04 Standard Stan Corn-Ref 6 30.76 2.806 6.40E+02 6.40E+02 N/A 30.76 N/A E06 Standard Stan Corn-Ref 7 33.65 1.903 8.00E+01 8.00E+01 N/A 33.65 N/A E08 Standard Stan Corn-Ref 8 35.89 1.000 1.00E+01 1.00E+01 N/A 35.89 N/A F02 Unknown B1 5 27.38 3.808 6.42E+03 5.94E+03 6.84E+02 27.49 1.57E-01 F04 Unknown B1 5 27.61 3.737 5.46E+03 5.94E+03 6.84E+02 27.49 1.57E-01 F06 Unknown B2 6 N/A N/A N/A N/A N/A N/A N/A F08 Unknown B2 6 29.96 2.985 9.65E+02 9.65E+02 N/A 29.96 N/A G02 Unknown B7 7 27.71 3.704 5.06E+03 4.90E+03 2.23E+02 27.75 6.18E-02 G04 Unknown B7 7 27.80 3.676 4.75E+03 4.90E+03 2.23E+02 27.75 6.18E-02 110
  8. 111 Gen tham chiếu: PCR Standard Curve Report with PCR Amp Cycle Graph PCR Base Line Subtracted Curve Fit Data Current Date: 11-Aug-05 04:02 PM Data generated on: 22-Jul-05 at 06:15 PM. Optical data file name: Data 22-Jul-05 1815-Truong-35S-dot 1-2.odm Plate Setup file used: GMO-35S.psm Protocol file used: GMO-35S.tmo Sample volume: 25.00 ul Hot Start? No W ell factor collection: Experimental Plate Comments Protocol Cycle 1: ( 1X) 95.0ºC Step 1: for 10:00 Cycle 2: ( 45X) 95.0ºC Step 1: for 00:15 60.0ºC Step 2: for 01:00 Data collection and real-time analysis enabled. Cycle 3: ( 1X) 4.0ºC Step 1: HOLD PCR Amp/Cycle Graph for FAM -490 Standard Curve Graph for FAM -490 111
  9. 112 Data Analysis Parameters Calculated threshold has been replaced by the user selected threshold 72.9. User selected baseline cycles are 2 to 20. Data analysis window is set at 95.00% of a cycle, centered at end of the cycle. Weighted Mean digital filtering has been applied. Global filtering is off. Standard Curve Spreadsheet Data for FAM -490 Units: copy number Type Identifier Rep Ct Log SQ SQ SQ Ct Ct Set SQ Mean SD Mean SD Point B02 Standard Stan Corn-Ref 1 28.55 4.612 4.10E+04 4.10E+04 N/A 28.55 N/A B04 Standard Stan Corn-Ref 2 30.62 3.709 5.12E+03 5.12E+03 N/A 30.62 N/A B06 Standard Stan Corn-Ref 3 33.22 2.806 6.40E+02 6.40E+02 N/A 33.22 N/A B08 Standard Stan Corn-Ref 4 35.84 1.903 8.00E+01 8.00E+01 N/A 35.84 N/A C02 Unknown B1 1 27.69 4.870 7.42E+04 9.61E+04 3.09E+04 27.42 3.86E-01 C04 Unknown B1 1 27.14 5.072 1.18E+05 9.61E+04 3.09E+04 27.42 3.86E-01 C06 Unknown B2 2 37.03 1.420 2.63E+01 8.38E+01 8.13E+01 36.04 1.40E+00 C08 Unknown B2 2 35.06 2.150 1.41E+02 8.38E+01 8.13E+01 36.04 1.40E+00 D02 Unknown B7 3 28.32 4.636 4.32E+04 4.35E+04 3.33E+02 28.32 9.02E-03 D04 Unknown B7 3 28.31 4.640 4.37E+04 4.35E+04 3.33E+02 28.32 9.02E-03 Đợt 6: Định lƣợng các mẫu thí nghiệm (lần 2) Promoter 35S: PCR Standard Curve Report with PCR Amp Cycle Graph PCR Base Line Subtracted Curve Fit Data Current Date: 11-Aug-05 05:04 PM Data generated on: 03-Aug-05 at 09:21 PM. Optical data file name: Data 03-Aug-05 2121 TAQMAN DOT 2-35S-MAU.odm Plate Setup file used: Taqman 35S dot 2 3-8-05.psm Protocol file used: GMO-35S.tmo Sample volume: 25.00 ul Hot Start? No W ell factor collection: Experimental Plate Comments Protocol 112
  10. 113 Cycle 1: ( 1X) 95.0ºC Step 1: for 10:00 Cycle 2: ( 45X) 95.0ºC Step 1: for 00:15 60.0ºC Step 2: for 01:00 Data collection and real-time analysis enabled. Cycle 3: ( 1X) 4.0ºC Step 1: HOLD PCR Amp/Cycle Graph for FAM -490 Standard Curve Graph for FAM -490 Data Analysis Parameters Calculated threshold has been replaced by the user selected threshold 65.7. User selected baseline cycles are 2 to 20. Data analysis window is set at 95.00% of a cycle, centered at end of the cycle. Weighted Mean digital filtering has been applied. Global filtering is off. Standard Curve Spreadsheet Data for FAM -490 Units: copy number Type Identifier Rep Ct Log SQ SQ SQ Ct Ct Set SQ Mean SD Mean SD Point E02 Standard St 35S 5-8 5 26.77 3.709 5.12E+03 5.12E+03 N/A 26.77 N/A E04 Standard St 35S 5-8 6 30.13 2.806 6.40E+02 6.40E+02 N/A 30.13 N/A E06 Standard St 35S 5-8 7 32.62 1.903 8.00E+01 8.00E+01 N/A 32.62 N/A E08 Standard St 35S 5-8 8 35.24 1.000 1.00E+01 1.00E+01 N/A 35.24 N/A F02 Unknown B4 8 26.35 3.920 8.32E+03 7.88E+03 6.32E+02 26.43 1.08E-01 F03 Unknown B4 8 26.51 3.871 7.43E+03 7.88E+03 6.32E+02 26.43 1.08E-01 F04 Unknown B1 9 26.60 3.842 6.95E+03 8.16E+03 1.71E+03 26.39 2.84E-01 F05 Unknown B1 9 26.19 3.972 9.38E+03 8.16E+03 1.71E+03 26.39 2.84E-01 F06 Unknown B2 10 28.93 3.085 1.22E+03 9.45E+02 3.85E+02 29.33 5.63E-01 F07 Unknown B2 10 29.73 2.828 6.73E+02 9.45E+02 3.85E+02 29.33 5.63E-01 F08 Unknown B6 11 34.16 1.395 2.48E+01 3.33E+01 1.20E+01 33.81 4.95E-01 F09 Unknown B6 11 33.46 1.622 4.18E+01 3.33E+01 1.20E+01 33.81 4.95E-01 113
  11. 114 F10 Unknown B7 12 27.36 3.594 3.93E+03 4.16E+03 3.22E+02 27.29 1.04E-01 F11 Unknown B7 12 27.21 3.642 4.39E+03 4.16E+03 3.22E+02 27.29 1.04E-01 G02 Unknown B10 13 26.05 4.019 1.05E+04 7.24E+03 4.56E+03 26.69 9.09E-01 G03 Unknown B10 13 27.33 3.603 4.01E+03 7.24E+03 4.56E+03 26.69 9.09E-01 G04 Unknown B11 14 24.99 4.362 2.30E+04 1.77E+04 7.47E+03 25.40 5.83E-01 G05 Unknown B11 14 25.81 4.095 1.25E+04 1.77E+04 7.47E+03 25.40 5.83E-01 Gen tham chiếu: PCR Standard Curve Report with PCR Amp Cycle Graph PCR Base Line Subtracted Curve Fit Data Current Date: 20-Aug-05 03:17 PM Data generated on: 03-Aug-05 at 09:21 PM. Optical data file name: Data 03-Aug-05 2121 TAQMAN DOT 2-GEN DC.odm Plate Setup file used: Taqman 35S dot 2 3-8-05.psm Protocol file used: GMO-35S.tmo Sample volume: 25.00 ul Hot Start? No W ell factor collection: Experimental Plate Comments Protocol Cycle 1: ( 1X) 95.0ºC Step 1: for 10:00 Cycle 2: ( 45X) 95.0ºC Step 1: for 00:15 60.0ºC Step 2: for 01:00 Data collection and real-time analysis enabled. Cycle 3: ( 1X) 4.0ºC Step 1: HOLD PCR Amp/Cycle Graph for FAM -490 114
  12. 115 Standard Curve Graph for FAM -490 Data Analysis Parameters Calculated threshold using the maximum correlation coefficient approach is 14.3. User selected baseline cycles are 2 to 19. Data analysis window is set at 95.00% of a cycle, centered at end of the cycle. Weighted Mean digital filtering has been applied. Global filtering is off. Standard Curve Spreadsheet Data for FAM -490 Units: copy number Type Identifier Rep Ct Log SQ SQ SQ Ct Ct Set SQ Mean SD Mean SD Point B02 Standard St CR 1-4 1 22.19 4.612 4.10E+04 4.10E+04 N/A 22.19 N/A B04 Standard St CR 1-4 2 24.81 3.709 5.12E+03 5.12E+03 N/A 24.81 N/A B06 Standard St CR 1-4 3 26.55 2.806 6.40E+02 6.40E+02 N/A 26.55 N/A B08 Standard St CR 1-4 4 30.57 1.903 8.00E+01 8.00E+01 N/A 30.57 N/A C02 Unknown B4 1 23.02 4.268 1.85E+04 1.29E+04 7.98E+03 23.63 8.57E-01 C03 Unknown B4 1 24.23 3.861 7.26E+03 1.29E+04 7.98E+03 23.63 8.57E-01 C04 Unknown B1 2 25.69 3.371 2.35E+03 2.59E+03 3.40E+02 25.57 1.70E-01 C05 Unknown B1 2 25.45 3.452 2.83E+03 2.59E+03 3.40E+02 25.57 1.70E-01 C06 Unknown B2 3 25.60 3.403 2.53E+03 4.46E+03 2.73E+03 25.00 8.47E-01 C07 Unknown B2 3 24.40 3.806 6.40E+03 4.46E+03 2.73E+03 25.00 8.47E-01 C08 Unknown B6 4 23.91 3.970 9.33E+03 9.27E+03 7.56E+01 23.92 1.05E-02 C09 Unknown B6 4 23.93 3.965 9.22E+03 9.27E+03 7.56E+01 23.92 1.05E-02 C10 Unknown B7 5 22.55 4.426 2.67E+04 1.52E+04 1.62E+04 23.82 1.79E+00 C11 Unknown B7 5 25.09 3.575 3.76E+03 1.52E+04 1.62E+04 23.82 1.79E+00 D02 Unknown B10 6 24.92 3.630 4.26E+03 4.27E+03 1.07E+01 24.92 3.23E-03 D03 Unknown B10 6 24.92 3.631 4.28E+03 4.27E+03 1.07E+01 24.92 3.23E-03 D04 Unknown B11 7 30.59 1.726 5.32E+01 3.31E+02 3.93E+02 29.01 2.23E+00 D05 Unknown B11 7 27.44 2.785 6.09E+02 3.31E+02 3.93E+02 29.01 2.23E+00 115
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