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

More documents

Recommendations

Info

Chapter 5Analyzing End-Point DataCluster VariationsThe SDS software graphs the results of an allelic discrimination run on a scatterplotcontrasting reporter dye fluorescence (Allele X R n versus Allele Y R n ). The softwarerepresents each well of the 384-well plate as a datapoint on the plot. The clustering ofthese datapoints can vary along the horizontal axis (Allele X), vertical axis(Allele Y), or diagonal (Allele X/Allele Y). This variation is due to differences in theextent of PCR amplification, which could result from differences in initial DNAconcentration.The example below shows the variation in clustering due to differences in the extentof PCR amplification.Allele Y homozygotesAllele X/Allele YheterozygotesOutliersAllele X homozygotesNo amplificationFigure 5-4Common Datapoint ClustersGenotypic Segregation of Datapoint ClustersFigure 5-4 illustrates the concept of genotypic segregation of samples within theallele plot. The plot contains four separate, distinct clusters that represent the NoTemplate Controls and the three possible genotypes (allele X homozygous, allele Yhomozygous, and heterozygous). Because of their homogenous genetic compliment,homozygous samples exhibit increased fluorescence along one axis of the plot(depending on the allele they contain). In contrast, heterozygous samples appearwithin the center of the plot because they contain copies of both alleles, and thereforeexhibit increased fluorescence for both reporter dyes.About OutliersSamples that did not cluster tightly may:• Contain rare sequence variations• Contain sequence duplications• Not contain a crucial reagent for amplification (the result of a pipetting error)5-8 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_End-Point.fm
Before You BeginBefore You BeginUsing the SDSSoftwareOnline HelpExamples inThis SectionFor specific instructions on any procedure described within this section, refer to theSequence Detection Systems Software Online Help. To get help at any time during theprocedure, click located within the dialog box or window in which you areworking.The illustrations and screenshots that appear within this chapter were created from aplate containing Pre-Developed TaqMan Assays and Reagents for AllelicDiscrimination run to screen 6 human genomic DNA samples (HD 1, 2, 3, 4, 7, 8) for2 targets (CYP 2C9*2 and CYP 2C19*2). Each well of the plate contains 1 µL DNA,TaqMan ® 2✕ Universal PCR Master Mix, forward and reverse primers, and FAM andVIC dye-labeled TaqMan probes.Figure 5-5 illustrates the arrangement of the assays, unknown samples, and notemplate control (NTC) wells on the plate.HD-1 HD-1HD-1HD-1HD-1HD-1HD-1 HD-1HD-1HD-1HD-2 HD-2HD-2HD-2HD-2HD-2HD-2 HD-2HD-2HD-2HD-3 HD-3HD-3HD-3HD-3HD-3HD-3 HD-3HD-3HD-3NTCHD-4 HD-4HD-4HD-4HD-4HD-4HD-4 HD-4HD-4HD-4HD-7 HD-7HD-7HD-7HD-7HD-7HD-7 HD-7HD-7HD-7HD-8 HD-8HD-8HD-8HD-8HD-8HD-8 HD-8HD-9HD-8Not in UseGR2107PDAR CYP 2C9*2PDAR CYP 2C19*2Figure 5-5Configuration of the Samples and Detectors on the Example PlateNote: The probes used in the example experiment were designed using the PrimerExpress ® Primer Design Software and by following the guidelines explained in“Assay Development Guidelines” on page B-2.IMPORTANT! The SDS software does not require that absolute quantification platescontain positive controls.Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 5-9DRAFTSeptember 1, 2004 11:39 am, CH_End-Point.fm
Page 1 and 2:
Applied Biosystems7900HT Fast Real-
Page 3 and 4:
ContentsPrefaceHow to Use This Guid
Page 5 and 6:
Chapter 4Operating the InstrumentNo
Page 7 and 8:
Chapter 7Maintaining the Instrument
Page 9 and 10:
Appendix D Theory of OperationFluor
Page 11 and 12:
PrefaceHow to Use This GuidePurpose
Page 13 and 14:
Safety and EMC Compliance Informati
Page 15 and 16:
Symbols on InstrumentsSymbols on In
Page 17 and 18:
General Instrument SafetyGeneral In
Page 19 and 20:
Chemical Waste SafetyChemical Safet
Page 21 and 22:
Physical Hazard SafetyOvervoltageRa
Page 23 and 24:
Workstation SafetyWorkstation Safet
Page 25 and 26:
Product Overview 11In This Chapter
Page 27 and 28:
7900HT FAST Real-Time PCR SystemSec
Page 29 and 30:
7900HT InstrumentInterchangeableThe
Page 31 and 32:
7900HT FAST Real-Time PCR SystemBar
Page 33 and 34:
Compatible ConsumablesCompatible Co
Page 35 and 36:
Instrument ConnectionsFixed-Positio
Page 37 and 38:
Section 1.2 Getting to Know the Sof
Page 39 and 40:
SDS Software Related Files and Form
Page 41 and 42:
7900HT FAST Real-Time PCR SystemMan
Page 43 and 44:
Esc F1 F2 F3 F4 F5 F6 F7 F8 F9 F10
Page 45 and 46:
Section 1.3 SDS Enterprise Database
Page 47 and 48:
* + Enter* +Enter3Down UpNumLockSys
Page 49 and 50:
About the SDS Enterprise Database S
Page 51 and 52:
Esc F1 F2 F3 F4 F5 F6 F7 F8 F9 F10
Page 53 and 54:
Getting Started 22In This Chapter G
Page 55 and 56:
Getting StartedUsing the SDSSoftwar
Page 57 and 58:
GR23967900HTPowering On the 7900HT
Page 59 and 60:
Using the SDS SoftwareUsing the SDS
Page 61 and 62:
Using the SDS SoftwareAbout theMess
Page 63 and 64:
Basic Software Skills TutorialNotes
Page 65 and 66:
Basic Software Skills TutorialExerc
Page 67 and 68:
Basic Software Skills TutorialExpor
Page 69 and 70:
Page 71 and 72:
Page 73 and 74:
Basic Software Skills TutorialLesso
Page 75 and 76:
Page 77 and 78:
Basic Software Skills Tutorial3. Se
Page 79 and 80:
Page 81 and 82:
Using SDS Plate DocumentsUsing SDS
Page 83 and 84:
Using SDS Plate DocumentsWell Inspe
Page 85 and 86:
Preparing a Run 33In This Chapter N
Page 87 and 88:
Notes for Database UsersDescription
Page 89 and 90:
Workflow OverviewWorkflow OverviewE
Page 91 and 92:
Workflow OverviewAllelicDiscriminat
Page 93 and 94:
Step 1 - Creating a Plate DocumentS
Page 95 and 96:
Step 2 - Applying Detectors and Mar
Page 97 and 98:
Page 99 and 100:
Page 101 and 102:
Step 3 - Configuring the Plate Docu
Page 103 and 104:
Step 5 - Programming the Plate Docu
Page 105 and 106:
Page 107 and 108:
Page 109 and 110:
Step 6 - Saving the Plate Document
Page 111 and 112:
Step 7 - Creating a Plate Document
Page 113 and 114:
Step 9 - Running the Plate on the 7
Page 115 and 116: Operating the Instrument 44In This
Page 117 and 118: Notes for Database UsersDescription
Page 119 and 120: Section 4.1 Consumable PreparationS
Page 121 and 122: Compatible ConsumablesTable 4-1Comp
Page 123 and 124: Preparing Optical Plates for Usea.
Page 125 and 126: Preparing TaqMan Low Density Arrays
Page 137 and 138: Section 4.2 Running an Individual P
Page 139 and 140: Running a Single Plate (Using the S
Page 141 and 142: Running a Single Plate (Using the S
Page 143 and 144: After the RunAfter the RunUser Acce
Page 145 and 146: Section 4.3 Automated OperationSect
Page 147 and 148: Operating the Software with an SDS
Page 149 and 150: Operating the Software without an S
Page 155 and 156: Running Plates Using the Automation
Page 157 and 158: Running Plates Using the Automation
Page 159 and 160: Analyzing End-Point Data 55In This
Page 161 and 162: Notes for Database UsersNotes for D
Page 163 and 164: Section 5.1 Allelic DiscriminationS
Page 165: OverviewTable 5-1Signal and Sequenc
Page 169 and 170: Opening the Run DataOpening the Run
Page 171 and 172: Calling and Scrutinizing Allelic Di
Page 177 and 178: After the AnalysisSaving theResults
Page 179 and 180: Analyzing Real-Time Data 66In This
Page 181 and 182: Notes for Database UsersDescription
Page 183 and 184: Section 6.1 Absolute Quantification
Page 185 and 186: OverviewAlgorithmicManipulation ofR
Page 187 and 188: Analysis ChecklistAnalysis Checklis
Page 189 and 190: Analyzing the DataAnalyzing the Dat
Page 191 and 192: Viewing ResultsViewing ResultsViewi
Page 193 and 194: Section 6.2 Relative Quantification
Page 195 and 196: OverviewAlgorithmic Manipulation of
Page 197 and 198: OverviewAutomatic Outlier RemovalAb
Page 199 and 200: Getting StartedGetting StartedUsing
Page 201 and 202: Options for Analyzing Relative Quan
Page 203 and 204: Creating the Studyd. In the Plate Q
Page 205 and 206: Analyzing the Study• Manual Ct -
Page 207 and 208: Analyzing the StudySpecifying the A
Page 209 and 210: Viewing ResultsViewing ResultsDispl
Page 211 and 212: Viewing ResultsAbout DownArrowsDown
Page 213 and 214: After the AnalysisAfter the Analysi
Page 215 and 216: Section 6.3 Dissociation Curve Anal
Page 217 and 218:
Before You BeginExample ResultsFigu
Page 219 and 220:
Opening the Run DataOpening the Run
Page 221 and 222:
Determining T m Values for the Anal
Page 223 and 224:
Section 6.4 Procedure ReferenceSect
Page 225 and 226:
Setting the Baseline and Threshold
Page 227 and 228:
Eliminating Outlying AmplificationE
Page 229 and 230:
Eliminating Outlying Amplification6
Page 231 and 232:
After the AnalysisSaving theResults
Page 233 and 234:
Maintaining the Instrument 77In Thi
Page 235 and 236:
Notes for Database UsersDescription
Page 237 and 238:
Section 7.1 Maintaining the 7900HT
Page 239 and 240:
Replacing the Sample BlockMaterials
Page 241 and 242:
Replacing the Sample Block9. Loosen
Page 243 and 244:
7900HT FAST Real-Time PCR SystemRep
Page 245 and 246:
Changing the Plate Adapter4. Place
Page 247 and 248:
Decontaminating the Sample BlockCle
Page 249 and 250:
Performing a Background RunPreparin
Page 251 and 252:
Performing a Background RunAnalyzin
Page 253 and 254:
Performing a Pure Dye RunAfter a ru
Page 255 and 256:
Performing a Pure Dye Run• Fast 9
Page 257 and 258:
Performing a Pure Dye RunAnalyzing
Page 259 and 260:
Adding Custom Dyes to the Pure Dye
Page 261 and 262:
Adding Custom Dyes to the Pure Dye
Page 263 and 264:
Verifying Instrument PerformanceTab
Page 265 and 266:
Verifying Instrument PerformancePre
Page 267 and 268:
Section 7.2 Maintaining the Plate H
Page 269 and 270:
Adjusting the Sensitivity of the Pl
Page 271 and 272:
Adjusting the Sensitivity of the Pl
Page 273 and 274:
7900HT FAST Real-Time PCR SystemAli
Page 275 and 276:
Aligning the Plate Handler5. Using
Page 277 and 278:
Aligning the Plate Handler7. Using
Page 279 and 280:
Aligning the Plate Handler13. Click
Page 281 and 282:
7900HT FAST Real-Time PCR SystemAli
Page 283 and 284:
Aligning the Fixed-Position Bar Cod
Page 285 and 286:
Section 7.3 Maintaining the Compute
Page 287 and 288:
Maintaining the SDS softwareSeveral
Page 289 and 290:
Troubleshooting 88In This Chapter T
Page 291 and 292:
Troubleshooting TableTable 8-1Troub
Page 293 and 294:
Low Precision or IrreproducibilityL
Page 295 and 296:
Low Precision or IrreproducibilityD
Page 297 and 298:
Background RunsBackground RunsBackg
Page 299 and 300:
Pure Dye Runs6. Repeat step 4 until
Page 301 and 302:
End-Point Runs (Allelic Discriminat
Page 303 and 304:
Software and 7900HT InstrumentTable
Page 305 and 306:
Zymark Twister Microplate Handler a
Page 307 and 308:
TaqMan Low Density ArrayTable 8-6Tr
Page 309 and 310:
Software ReferenceAAIn This Appendi
Page 311 and 312:
Importing Plate Document Setup Tabl
Page 313 and 314:
Setup Table Files1. File VersionDes
Page 315 and 316:
Setup Table Files6. Detectors ListD
Page 317 and 318:
Setup Table Files9. Markers List (A
Page 319 and 320:
Setup Table FilesFormat(for a singl
Page 321 and 322:
Setup Table FilesFormat(for a singl
Page 323 and 324:
Setup Table FilesExample Code A-16A
Page 325 and 326:
Exporting Plate Document Data4. In
Page 327 and 328:
Operating the SDS Software from a C
Page 329 and 330:
Using the Search ToolLogical Operat
Page 331 and 332:
Connecting SDS Software to the Data
Page 333 and 334:
Designing TaqMan Reagent-BasedAssay
Page 335 and 336:
Assay Development Guidelines• For
Page 337 and 338:
Design Tips for Allelic Discriminat
Page 339 and 340:
Kits, Reagents, and ConsumablesCCIn
Page 341 and 342:
Consumables and DisposablesConsumab
Page 343 and 344:
Instrument Maintenance and Verifica
Page 345 and 346:
Custom Oligonucleotide SynthesisTab
Page 347 and 348:
Theory of OperationDDIn This Append
Page 349 and 350:
Fluorescent-Based ChemistriesBasics
Page 351 and 352:
Real-Time Data AnalysisPhase 2: Lin
Page 353 and 354:
Real-Time Data AnalysisSignificance
Page 355 and 356:
A similar equation for the endogeno
Page 357 and 358:
Limited Warranty StatementEEWarrant
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
show all

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

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?