Applied Biosystems 7900HT Fast Real-Time PCR System and SDS ...

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Appendix DTheory of OperationFluorescence vs.AmplifiedProductWhen using TaqMan fluorogenic probes with the 7900HT instrument, fluorescenceemission increases in direct proportion to the amount of specific amplified product.As Figure D-4 on page D-5 demonstrates, the graph of normalized reporter (R n ) vs.cycle number during PCR appears to have three stages. Initially, R n appears as a flatline because the fluorescent signal is below the detection limit of the 7900HTinstrument. In the second stage, the signal can be detected as it continues to increasein direct proportion to the increase in the products of PCR. As PCR productcontinues to increase, the ratio of AmpliTaq Gold polymerase to PCR productdecreases. When template concentration reaches 10 -8 M, PCR product ceases to growexponentially. This signals the third stage of R n change, which is roughly linear andfinally reaches a plateau at about 10 -7 M (Martens and Naes, 1989).The progressive cleavage of TaqMan fluorescent probes during the PCR makespossible the correlation between initial template concentration and the rise influorescence. As the concentration of amplified product increases in a sample, sodoes the R n value. During the exponential growth stage (the geometric phase), therelationship of amplified PCR product to initial template can be shown in thefollowing equation:N c= N1 ( + E) cwhere N c is the concentration of amplified product at any cycle, N is the initialconcentration of target template, E is the efficiency of the system, and c is the cyclenumber.CalculatingThreshold CyclesThe Applied Biosystems 7900HT Fast Real-Time PCR System creates quantifiablerelationships between test samples based on the number of cycles elapsed beforeachieving detectable levels of fluorescence. Test samples containing a greater initialtemplate number cross the detection threshold at a lower cycle than samplescontaining lower initial template. The SDS software uses a Threshold setting todefine the level of detectable fluorescence.The threshold cycle (C T ) for a given amplification curve occurs at the point that thefluorescent signal grows beyond the value of the threshold setting. The C T representsa detection threshold for the 7900HT instrument and is dependent on two factors:• Starting template copy number• Efficiency of DNA amplification the PCR systemHow the SDS Software Determines C T sTo determine the C T for an Amplification plot, the SDS software uses data collecteddata from a predefined range of PCR cycles called the ‘baseline’ (the defaultbaseline occurs between cycles 3 and 15). First, the software calculates amathematical trend based on the baseline cycles’ R n values to generate a baselinesubtracted Amplification plot of ∆R n versus cycle number. Next, an algorithmsearches for the point on the Amplification plot at which the ∆R n value crosses thethreshold setting (the default threshold setting is 0.2). The fractional cycle at whichthe intersection occurs is defined as the threshold cycle (C T ) for the plot.Note: It may be necessary to adjust the baseline and threshold settings to obtainaccurate and precise data. For further information on resetting the baseline andthreshold settings, see “Setting the Baseline and Threshold Values” on page 6-46.D-6 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, App_Theory.fm
Real-Time Data AnalysisSignificance ofThreshold CyclesBeginning with the equation describing the exponential amplification of the PCR:X n = X m ( 1 + E X ) n – mwhere:X n = number of target molecules at cycle n (so that n ≥ m)X m = number of target molecules at cycle m (so that m ″ n)E X = efficiency of target amplification (between 0–1)n - m = number of cycles elapsed between cycle m and cycle nAmplicons designed and optimized according to Applied Biosystems guidelines(amplicon size
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Applied Biosystems7900HT Fast Real-
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ContentsPrefaceHow to Use This Guid
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Chapter 4Operating the InstrumentNo
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Chapter 7Maintaining the Instrument
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Appendix D Theory of OperationFluor
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PrefaceHow to Use This GuidePurpose
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Safety and EMC Compliance Informati
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Symbols on InstrumentsSymbols on In
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General Instrument SafetyGeneral In
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Chemical Waste SafetyChemical Safet
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Physical Hazard SafetyOvervoltageRa
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Workstation SafetyWorkstation Safet
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Product Overview 11In This Chapter
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7900HT FAST Real-Time PCR SystemSec
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7900HT InstrumentInterchangeableThe
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7900HT FAST Real-Time PCR SystemBar
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Compatible ConsumablesCompatible Co
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Instrument ConnectionsFixed-Positio
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Section 1.2 Getting to Know the Sof
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SDS Software Related Files and Form
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7900HT FAST Real-Time PCR SystemMan
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Esc F1 F2 F3 F4 F5 F6 F7 F8 F9 F10
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Section 1.3 SDS Enterprise Database
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* + Enter* +Enter3Down UpNumLockSys
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About the SDS Enterprise Database S
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Esc F1 F2 F3 F4 F5 F6 F7 F8 F9 F10
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Getting Started 22In This Chapter G
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Getting StartedUsing the SDSSoftwar
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GR23967900HTPowering On the 7900HT
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Using the SDS SoftwareUsing the SDS
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Using the SDS SoftwareAbout theMess
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Basic Software Skills TutorialNotes
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Basic Software Skills TutorialExerc
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Basic Software Skills TutorialExpor
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Basic Software Skills TutorialLesso
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Basic Software Skills Tutorial3. Se
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Using SDS Plate DocumentsUsing SDS
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Using SDS Plate DocumentsWell Inspe
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Preparing a Run 33In This Chapter N
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Notes for Database UsersDescription
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Workflow OverviewWorkflow OverviewE
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Workflow OverviewAllelicDiscriminat
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Step 1 - Creating a Plate DocumentS
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Step 2 - Applying Detectors and Mar
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Step 3 - Configuring the Plate Docu
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Step 5 - Programming the Plate Docu
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Step 6 - Saving the Plate Document
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Step 7 - Creating a Plate Document
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Step 9 - Running the Plate on the 7
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Operating the Instrument 44In This
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Section 4.1 Consumable PreparationS
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Compatible ConsumablesTable 4-1Comp
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Preparing Optical Plates for Usea.
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Preparing TaqMan Low Density Arrays
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Section 4.2 Running an Individual P
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Running a Single Plate (Using the S
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Running a Single Plate (Using the S
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After the RunAfter the RunUser Acce
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Section 4.3 Automated OperationSect
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Operating the Software with an SDS
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Operating the Software without an S
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Running Plates Using the Automation
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Running Plates Using the Automation
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Analyzing End-Point Data 55In This
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Notes for Database UsersNotes for D
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Section 5.1 Allelic DiscriminationS
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OverviewTable 5-1Signal and Sequenc
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Before You BeginBefore You BeginUsi
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Opening the Run DataOpening the Run
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Calling and Scrutinizing Allelic Di
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After the AnalysisSaving theResults
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Analyzing Real-Time Data 66In This
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Section 6.1 Absolute Quantification
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OverviewAlgorithmicManipulation ofR
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Analysis ChecklistAnalysis Checklis
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Analyzing the DataAnalyzing the Dat
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Viewing ResultsViewing ResultsViewi
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Section 6.2 Relative Quantification
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OverviewAlgorithmic Manipulation of
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OverviewAutomatic Outlier RemovalAb
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Getting StartedGetting StartedUsing
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Options for Analyzing Relative Quan
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Creating the Studyd. In the Plate Q
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Analyzing the Study• Manual Ct -
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Analyzing the StudySpecifying the A
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Viewing ResultsViewing ResultsDispl
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Viewing ResultsAbout DownArrowsDown
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After the AnalysisAfter the Analysi
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Section 6.3 Dissociation Curve Anal
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Before You BeginExample ResultsFigu
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Opening the Run DataOpening the Run
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Determining T m Values for the Anal
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Section 6.4 Procedure ReferenceSect
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Setting the Baseline and Threshold
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Eliminating Outlying AmplificationE
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Eliminating Outlying Amplification6
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After the AnalysisSaving theResults
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Maintaining the Instrument 77In Thi
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Section 7.1 Maintaining the 7900HT
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Replacing the Sample BlockMaterials
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Replacing the Sample Block9. Loosen
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7900HT FAST Real-Time PCR SystemRep
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Changing the Plate Adapter4. Place
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Decontaminating the Sample BlockCle
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Performing a Background RunPreparin
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Performing a Background RunAnalyzin
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Performing a Pure Dye RunAfter a ru
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Performing a Pure Dye Run• Fast 9
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Performing a Pure Dye RunAnalyzing
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Adding Custom Dyes to the Pure Dye
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Adding Custom Dyes to the Pure Dye
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Verifying Instrument PerformanceTab
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Verifying Instrument PerformancePre
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Section 7.2 Maintaining the Plate H
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Adjusting the Sensitivity of the Pl
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Adjusting the Sensitivity of the Pl
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7900HT FAST Real-Time PCR SystemAli
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Aligning the Plate Handler5. Using
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Aligning the Plate Handler7. Using
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Aligning the Plate Handler13. Click
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7900HT FAST Real-Time PCR SystemAli
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Aligning the Fixed-Position Bar Cod
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Section 7.3 Maintaining the Compute
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Maintaining the SDS softwareSeveral
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Troubleshooting 88In This Chapter T
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Troubleshooting TableTable 8-1Troub
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Low Precision or IrreproducibilityL
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Low Precision or IrreproducibilityD
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Background RunsBackground RunsBackg
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Pure Dye Runs6. Repeat step 4 until
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Page 329 and 330: Using the Search ToolLogical Operat
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Page 333 and 334: Designing TaqMan Reagent-BasedAssay
Page 335 and 336: Assay Development Guidelines• For
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Page 341 and 342: Consumables and DisposablesConsumab
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Page 347 and 348: Theory of OperationDDIn This Append
Page 349 and 350: Fluorescent-Based ChemistriesBasics
Page 351: Real-Time Data AnalysisPhase 2: Lin
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Page 359 and 360: BibliographyGeneral Paperson TaqMan
Page 361 and 362: Klein, D., Janda, P., Steinborn, R.
Page 363 and 364: Dolken, L., F. , Schuler, and G. ,
Page 365 and 366: AllelicDiscriminationVirtanen M., H
Page 367 and 368: Glossary.sdd.sdm.sds.sdt∆C T∆
Page 369 and 370: ackgroundbaselinecalibratorcDNA(com
Page 371 and 372: minor groovebinder (MGB)multicompon
Page 373 and 374: R nrunsessionsingle nucleotidepolym
Page 375 and 376: IndexSymbols- 6-31↑ 6-33Numerics7
Page 377 and 378: fluorescent, common sources 8-7isol
Page 379 and 380: adjusting step parameters 3-21confi
Page 381 and 382: sample block locking bar 7-8sample
Page 384: Worldwide Sales and SupportApplied
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