User Bulletin Applied Biosystems 3400 DNA Synthesizer April 2007 SUBJECT: New Features in 3400 DNA Synthesizer Firmware v1.4.1 In This User Bulletin This user bulletin covers: Overview...........................................................2 Changes to the 1.0-µm Scale Default Synthesis Cycle Scripts..................2 Changes to the Calculation of Trityl Monitor ASWY.........................2 New Output in the Run Report..........................................4
New Features in 3400 DNA Synthesizer Firmware v1.4.1 Overview This user bulletin describes new features available in 3400 DNA Synthesizer firmware v1.4.1. These features are: Changes to the 1.0-µm Scale Default Synthesis Cycle Scripts Changes to the Calculation of Trityl Monitor ASWY New Output in the Run Report For more information about the 3400 DNA Synthesizer, see the Applied Biosystems 3400 DNA Synthesizer User Guide (PN 4334680). Changes to the 1.0-µm Scale Default Synthesis Cycle Scripts The v1.4.1 firmware adds additional ACN and Flush to Waste steps to the default 1µm-PO, 1µm-PS, and 1µm-RNA synthesis cycle scripts. These steps enhance oligonucleotide quality when different sequences are synthesized during a single synthesis run. Note: RNA synthesis is not supported by Applied Biosystems. Please contact your Applied Biosystems Technical Support representative for more information. Changes to the Calculation of Trityl Monitor ASWY The v1.4.1 firmware now sets the Average Step-wise Yield (ASWY) to 100% when the firmware encounters a new maximum peak area, following an established minimum area. The ASWY is displayed on the front of the 3400 DNA Synthesizer. The new algorithm calculates the ASWY for each base, using this equation: Peak Area ASWY = min a ------------------------------ 100% Peak Area max where Peak Area min and Peak Area max are the lowest and highest peak areas for the run so far, and a = (base number) 1. When the firmware encounters a new Peak Area max, the firmware assumes the base coupling proceeded at the maximum possible efficiency and sets the ASWY to 100%. An example is given in Table 1, below. 2 3400 DNA Synthesizer User Bulletin
Changes to the Calculation of Trityl Monitor ASWY Table 1 shows an example of ASWY and the values used to calculate it for a 9-base sequence. The peak area for Base 3 is larger than the Base 2 peak area (the previously established maximum), so ASWY is reset to 100% (indicated in red) and the Peak Area max is updated. Table 1 Representative ASWY values for a 9-base sequence Base Peak Area Peak Area min Peak Area max ASWY 1 429.8 2 364.8 364.8 364.8 100.0 3 443.9 364.8 443.9 100.0 4 414.2 364.8 443.9 93.7 5 398.1 364.8 443.9 95.2 6 440.9 364.8 443.9 96.2 7 389.6 364.8 443.9 96.8 8 375.9 364.8 443.9 97.2 9 357.1 357.1 443.9 97.3 The data for Base 1 reflects detritylation of the base originally attached to the solid synthesis support. Because the first detritylation peak area can be unreliable, the ASWY is not calculated for Base 1. Also, the Base 1peak area is omitted when determining the Peak Area min and Peak Area max values. A Note About ASWY and Overall Yield While overall ASWY by trityl monitoring is useful as a superficial indicator of oligonucleotide quality, it is not intended to be a precise measure of coupling yield; instead it is better used for early detection and diagnosis of instrument-related problems. Examine peak area values in the Run Report to look for anomalous trends before assessing oligonucleotide quality when evaluating overall ASWY by trityl monitoring. Applied Biosystems recommends that you determine overall coupling yield using an appropriate direct analysis method such as RP-HPLC, HPCE, mass spectrometry, or gel electrophoresis, particularly for new or novel oligonucleotides. Such analysis can provide additional assurance of desired results over any single measurement method. 3400 DNA Synthesizer User Bulletin 3
New Features in 3400 DNA Synthesizer Firmware v1.4.1 New Output in the Run Report Three new items have been added to the Run Report. See the example run report on page 5. Final DMT Status Cleavage Status Final Peak Area Values for Trityl-Off Syntheses The Run Report now indicates if DMT was removed as the last step of the synthesis. DMT removal is indicated by: Final DMT Off=Yes (also referred to as Trityl Off synthesis) for a synthesis where the DMT was removed or Final DMT Off=No (also referred to as Trityl On synthesis) for a synthesis where the DMT was not removed This information appears for each column in the Sequences section of the Run Report. The Run Report now indicates if the completed synthesis is subsequently cleaved from its solid support while remaining on the instrument. Cleavage status is indicated by: Cleavage=Yes for an oligonucleotide cleaved from the solid support or Cleavage=No for an oligonucleotide that remained affixed to the solid support This information appears at the end the entry for each column in the Sequences section of the Run Report. For all trityl-off syntheses, the peak area and peak height values for the final base detritylation are now included in the Trityl Results section of the Run Report. (Prior versions of the Run Report did not include information for the final base.) 4 3400 DNA Synthesizer User Bulletin
New Output in the Run Report Example Run Report Run Title: 03-01-07 Start Time: 2007-03-01 14:36:20 PST End Time: 2007-03-01 19:11:46 PST Run Time: 4h, 35m, 26s Instrument: ab3400-56 Software: AB 3400 DNA Synthesizer 1.4.1 Cycle: 1um-PO ================================== AB 3400 DNA Synthesizer Run Report ================================== ========= Sequences ========= Column 1: 25-mer-FC (Size=25; MW=7641; Tm=59.3; Final DMT Off=Yes; Cleavage=Yes) 5'> TCA TCA AAG CAT GCA TGG CCG TGC T <3' Column 2: AT-25 (Size=25; MW=7659; Tm=38.0; Final DMT Off=Yes; Cleavage=Yes) 5'> ATA TAT ATA TAT ATA TAT ATA TAT A <3' Column 3: 25-mer-FC (Size=25; MW=7641; Tm=59.3; Final DMT Off=Yes; Cleavage=Yes) 5'> TCA TCA AAG CAT GCA TGG CCG TGC T <3' Column 4: AT-25 (Size=25; MW=7659; Tm=38.0; Final DMT Off=Yes; Cleavage=Yes) 5'> ATA TAT ATA TAT ATA TAT ATA TAT A <3' ============== Trityl Results ============== Trityl levels were monitored until they fell within 5% of baseline Minimum acceptable step-wise yield was 80% Average step-wise yield for column 1 was 98.1% Average step-wise yield for column 2 was 97.9% Average step-wise yield for column 3 was 97.7% Average step-wise yield for column 4 was 97.7% Base Peak Area Peak Height Step No. Col 1 Col 2 Col 3 Col 4 Col 1 Col 2 Col 3 Col 4 Time -----+--------------------------------+--------------------------------+----- 1 746.4 721.7 584.4 735.0 30.79 30.77 23.22 34.55 40s 2 793.2 731.0 620.5 849.5 28.99 33.28 25.01 27.37 48s 3 623.3 625.1 399.4 746.3 32.77 33.21 22.71 30.43 45s 4 641.7 720.8 447.0 738.5 30.67 35.38 26.57 26.06 45s 5 542.2 714.9 360.9 646.4 29.01 35.55 26.32 34.90 41s 6 606.3 680.6 466.8 596.6 30.67 38.29 22.59 27.74 44s 7 586.3 718.2 417.5 584.6 30.52 28.23 23.03 29.86 44s 8 616.9 650.8 437.2 810.1 26.96 31.50 18.14 21.81 52s 9 620.0 709.2 399.1 599.0 23.39 27.28 19.36 37.00 42s 10 661.4 596.3 444.3 661.5 26.27 32.14 25.01 28.74 48s 11 506.2 654.6 402.6 629.6 28.50 26.93 23.61 31.33 45s 12 669.8 541.5 424.0 694.5 23.15 28.74 22.86 27.03 49s 13 631.5 589.3 356.9 581.0 23.00 25.42 17.80 26.50 47s 14 740.4 858.2 406.4 587.8 21.00 21.12 21.66 29.21 50s 15 704.4 764.1 472.0 577.8 19.85 23.42 20.56 26.54 50s 16 666.9 734.5 447.3 490.1 21.46 23.88 21.37 25.66 50s 17 528.2 657.1 409.4 632.5 21.44 27.18 14.36 23.76 52s 18 682.2 753.1 411.8 659.3 21.12 20.83 15.48 23.93 52s 19 638.6 632.2 407.2 761.5 22.86 25.64 16.04 20.51 54s 20 596.7 835.1 351.8 779.1 21.07 18.58 15.24 20.39 57s 21 614.6 728.3 408.1 679.5 21.93 20.07 16.80 20.83 55s 22 651.1 658.9 432.2 779.8 20.37 22.54 15.85 21.64 53s 23 601.4 702.9 410.3 782.0 22.64 17.78 17.68 18.00 59s 24 603.0 520.8 426.3 728.4 19.36 20.73 13.50 18.05 57s 25 652.8 538.0 375.5 568.5 22.83 19.27 14.77 19.88 54s 3400 DNA Synthesizer User Bulletin 5
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