ABI Prism® 7900HT Sequence Detection System ... - OpenWetWare

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Theory of Operation A In This Appendix This appendix discusses the following topics: A Topic See Page Fluorescent-Based Chemistries A-2 Fluorescence <strong>Detection</strong> and Data Collection A-4 Mathematical Transformations A-5 Real-TimeDataAnalysis A-7 Theory of Operation A-1
Fluorescent-Based Chemistries Fundamentals of the 5´ Nuclease Assay A-2 Theory of Operation The PCR reaction exploits the 5´ nuclease activity of AmpliTaq Gold ® DNA Polymerase to cleave a TaqMan ® probe during PCR. The TaqMan probe contains a reporter dye at the 5´ end of the probe and a quencher dye at the 3´ end of the probe. During the reaction, cleavage of the probe separates the reporter dye and the quencher dye, which results in increased fluorescence of the reporter. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye. The figure below shows the forklike-structure-dependent, polymerization-associated 5´–3´ nuclease activity of AmpliTaq Gold DNA Polymerase during PCR. Polymerization 5' 3' 5' Foward Primer Strand displacement R R R Probe When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence primarily by Förster-type energy transfer (Förster, 1948; Lakowicz, 1983). During PCR, if the target of interest is present, the probe specifically anneals between the forward and reverse primer sites. The 5´–3´ nucleolytic activity of the AmpliTaq Gold DNA Polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target. The probe fragments are then displaced from the target, and polymerization of the strand continues. The 3´ end of the probe is blocked to prevent extension of the probe during PCR. This process occurs in every cycle and does not interfere with the exponential accumulation of product. The increase in fluorescence signal is detected only if the target sequence is complementary to the probe and is amplified during PCR. Because of these requirements, any nonspecific amplification is not detected. Q 3' R = Reporter Q = Quencher Reverse Primer 5' Probe 3' 3' 5' 5' 3' 5' Cleavage 5' Probe 3' 3' 5' 5' 3' 5' Polymerization completed Q Probe R 5' 3' 5' 5' 3' 5' Q Q 3' 5' 3' 5'
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ABI PRISM ® 7900HT Sequence Detect
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© Copyright 2001, Applied Biosyste
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iv Section:PlateDocumentSetup......
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vi A Theory of Operation Fluorescen
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Attention Words and Warning Labels
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About Waste Profiles A waste profil
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Safe Instrument Use Before Operatin
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Product Overview 2 In This Chapter
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Section: Getting to Know the Hardwa
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Interchangeable Thermal Cycler Bloc
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Computer Description The computer c
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Zymark Twister Microplate Handler D
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Instrument Connections Electrical C
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Section: Getting to Know the Softwa
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Managing Sequence Detection System
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Getting Started 3 In This Chapter T
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Using the SDS Software Online Help
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Turning on the ABI PRISM 7900HT Seq
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Using the SDS Software Workspace La
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Using the General Toolbar Using the
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Basic Software Skills Tutorial Abou
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Exercise 3: Opening a Plate Documen
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Lesson 2: Viewing and Resizing Pane
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Exercise 2: Selecting Multiple Well
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Lesson 4: Using the Hand-Held Bar C
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Using SDS Plate Documents Using Mul
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Run Setup and Basic Operation 4 In
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Setup Checklists Experiments/Runs P
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Section: Plate Document Setup In Th
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Step 2 - Applying Detectors and Mar
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Creating an Allelic Discrimination
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Step 3 - Configuring the Plate Docu
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Step 4 - Programming the Plate Docu
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To create a method for the absolute
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Step 5 - Saving the Plate Document
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Step 7 - Applying Sample and Plate
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Section: Running an Individual Plat
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Preparing and Running a Single Plat
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Operating the 7900HT Instrument Usi
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After the Run Analyzing the Run Dat
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Section: Running Multiple Plates Us
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Adding a Plate Document to the Plat
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To create plate documents from a te
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Adding Plates to the Plate Queue Re
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Loading Plates To load plates onto
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End-Point Analysis 5 In This Chapte
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Section: Allelic Discrimination In
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Algorithmic Manipulation of Raw All
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Before You Begin Using SDS Online H
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Analyzing a Completed Allelic Discr
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Calling Allele Types To call allele
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Scrutinizing the Allele Calls To an
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After the Analysis Changing the Pla
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Real-Time Runs on the 7900HT Instru
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Overview About Absolute Quantificat
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Before You Begin Using SDS Online H
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Analyzing the Run Data Configuring
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Setting the Baseline and Threshold
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Eliminating Outliers For any PCR, e
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Viewing the Standard Curve 6-14 Rea
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6-16 Real-Time Analysis
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Overview About Dissociation Curve A
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Analysis Checklist WhereYouArein th
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Determining T m Values for the Anal
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After the Analysis Changing the Pla
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Recommended Maintenance Schedule Ma
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Replacing the Sample Block When to
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7-6 System Maintenance To remove th
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7-8 System Maintenance To replace t
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7-10 System Maintenance To replace
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Cleaning the Sample Block Wells 7-1
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Prepare a Background Plate Document
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Extracting the Background 7-16 Syst
Page 149 and 150: Materials Required The following ma
Page 151 and 152: Extracting Pure Dye Information fro
Page 153 and 154: Constructing a Custom Pure Dye Plat
Page 155 and 156: Verifying Instrument Performance Us
Page 157 and 158: Preparing and Running an RNase P Pl
Page 159 and 160: Adjusting the Sensitivity of the Pl
Page 161 and 162: Testing the Adjustment 7-30 System
Page 163 and 164: Aligning the Plate Handler When to
Page 165 and 166: 7-34 System Maintenance To align th
Page 167 and 168: Re-checking the Input Stack 1 7-36
Page 169 and 170: Defining the Positions of the Remai
Page 171 and 172: Aligning the Fixed-Position Bar Cod
Page 173 and 174: 7-42 System Maintenance To position
Page 175 and 176: 7-44 System Maintenance
Page 177 and 178: General Computer Maintenance Mainte
Page 179 and 180: Maintaining the SDS software Admini
Page 181 and 182: Troubleshooting Table 8-2 Troublesh
Page 183 and 184: Low Precision or Irreproducibility
Page 185 and 186: Writing on the Reaction Plates Fluo
Page 187 and 188: Background Runs Background Troubles
Page 189 and 190: Pure Dye Runs Pure Dye Troubleshoot
Page 191 and 192: 8-12 Troubleshooting Troubleshootin
Page 193 and 194: Software and 7900HT Instrument Trou
Page 195 and 196: 8-16 Troubleshooting Troubleshootin
Page 198: User Bulletins 9 9 About This Chapt
Page 203 and 204: Fluorescence Detection and Data Col
Page 205 and 206: Normalization of Reporter Signals A
Page 207 and 208: Determining Initial Template Concen
Page 209 and 210: Calculating Threshold Cycles A-10 T
Page 212 and 213: Importing and Exporting Plate Docum
Page 214 and 215: Configuring the Setup Table File wi
Page 216 and 217: About the Setup Table File Format S
Page 218 and 219: Setup Table Elements (continued) Nu
Page 220 and 221: Exporting Plate Document Data Expor
Page 222 and 223: Designing TaqMan Assays C In This A
Page 224 and 225: Design Probes and Primers The follo
Page 226 and 227: Design Tips for Allelic Discriminat
Page 228 and 229: Kits, Reagents and Consumables D In
Page 230 and 231: Consumables and Disposables The ABI
Page 232: TaqMan Pre-Developed Assays and Rea
Page 236: Contacting Technical Support F Serv
Page 239 and 240: G-2 Limited Warranty Statement No a
Page 241 and 242: A(continued) Automation Controller
Page 243 and 244: I(continued) installing plate adapt
Page 245 and 246: Q quantifying probes and primers C-
Page 248 and 249: Headquarters 850 Lincoln Centre Dri
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