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

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Section: Allelic Discrimination In This Section This section contains the following information: Topic See Page Overview 5-4 Before You Begin 5-7 Analysis Checklist 5-8 Analyzing a Completed Allelic Discrimination Run 5-9 Calling and Scrutinizing Allelic Discrimination Data 5-10 After the Analysis 5-15 End-Point Analysis 5-3
Overview Allelic Discrimination on the 7900HT Instrument Employing the 5´ Nuclease Assay for Allelic Discrimination 5-4 End-Point Analysis The ABI PRISM 7900HT Sequence Detection System supports allelic discrimination using TaqMan ® probes. Allelic discrimination is the process by which two variants of a single nucleic acid sequence are detected in a prepared sample. Allelic discrimination chemistry can be used for single-nucleotide polymorphism (SNP) detection. Allelic discrimination on the 7900HT instrument is made possible through the use of the fluorogenic 5´ nuclease assay (see page A-2). During the PCR, the fluorogenic probes anneal specifically to complementary sequences between the forward and reverse primer sites on the template DNA. Then during extension, AmpliTaq Gold® DNA polymerase cleaves the probes hybridized to the matching allele sequence(s) present in each sample. The cleavage of each matched probe separates the reporter dye from the quencher dye, which results in increased fluorescence by the reporter. After thermal cycling, the plate is run on the 7900HT instrument which reads the fluorescence generated during the PCR amplification. By quantifying and comparing the fluorescent signals using the SDS software, it is possible to determine the allelic content of each sample on the plate. Mismatches between a probe and target reduce the efficiency of probe hybridization. Furthermore, AmpliTaq Gold DNA polymerase is more likely to displace the mismatched probe than to cleave it, releasing the reporter dye. By running the extension phase of the PCR at the optimal annealing temperature for the probes, the lower melting temperatures (T m ) for mismatched probes minimizes their cleavage and consequently their fluorescent contribution. The figure below illustrates results from matches and mismatches between target and probe sequences in TaqMan ® PDARs for AD assays (Livak et al., 1995;Livaket al., 1999). Allele X V Probe-target sequence Match match Probe-target sequence Mismatch mismatch (higher Tm ) Allele Y F The table below shows the correlation between fluorescence signals and sequences present in the sample. A substantial increase in… Indicates… VIC fluorescence only homozygosity for Allele X. FAM fluorescence only homozygosity for Allele Y. both fluorescent signals heterozygosity. Q Q F Q Probe-target sequence Match match Probe-target sequence Mismatch mismatch (higher Tm) V Q V F Q Legend VIC FAM Quencher AmpliTaq Gold DNA Polymerase GR1556
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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
Page 44 and 45: Exercise 3: Opening a Plate Documen
Page 46 and 47: Lesson 2: Viewing and Resizing Pane
Page 48 and 49: Exercise 2: Selecting Multiple Well
Page 50 and 51: Lesson 4: Using the Hand-Held Bar C
Page 52 and 53: Using SDS Plate Documents Using Mul
Page 54 and 55: Run Setup and Basic Operation 4 In
Page 56 and 57: Setup Checklists Experiments/Runs P
Page 58 and 59: Section: Plate Document Setup In Th
Page 60 and 61: Step 2 - Applying Detectors and Mar
Page 62 and 63: Creating an Allelic Discrimination
Page 64 and 65: Step 3 - Configuring the Plate Docu
Page 66 and 67: Step 4 - Programming the Plate Docu
Page 68 and 69: To create a method for the absolute
Page 70 and 71: Step 5 - Saving the Plate Document
Page 72 and 73: Step 7 - Applying Sample and Plate
Page 74 and 75: Section: Running an Individual Plat
Page 76 and 77: Preparing and Running a Single Plat
Page 78 and 79: Operating the 7900HT Instrument Usi
Page 80 and 81: After the Run Analyzing the Run Dat
Page 82 and 83: Section: Running Multiple Plates Us
Page 84 and 85: Adding a Plate Document to the Plat
Page 86 and 87: To create plate documents from a te
Page 88 and 89: Adding Plates to the Plate Queue Re
Page 90 and 91: Loading Plates To load plates onto
Page 92 and 93: End-Point Analysis 5 In This Chapte
Page 96 and 97: Algorithmic Manipulation of Raw All
Page 98 and 99: Before You Begin Using SDS Online H
Page 100 and 101: Analyzing a Completed Allelic Discr
Page 102 and 103: Calling Allele Types To call allele
Page 104 and 105: Scrutinizing the Allele Calls To an
Page 106: After the Analysis Changing the Pla
Page 109 and 110: Real-Time Runs on the 7900HT Instru
Page 111 and 112: Overview About Absolute Quantificat
Page 113 and 114: Before You Begin Using SDS Online H
Page 115 and 116: Analyzing the Run Data Configuring
Page 117 and 118: Setting the Baseline and Threshold
Page 119 and 120: Eliminating Outliers For any PCR, e
Page 121 and 122: Viewing the Standard Curve 6-14 Rea
Page 123 and 124: 6-16 Real-Time Analysis
Page 125 and 126: Overview About Dissociation Curve A
Page 127 and 128: Analysis Checklist WhereYouArein th
Page 129 and 130: Determining T m Values for the Anal
Page 131 and 132: After the Analysis Changing the Pla
Page 133 and 134: Recommended Maintenance Schedule Ma
Page 135 and 136: Replacing the Sample Block When to
Page 137 and 138: 7-6 System Maintenance To remove th
Page 139 and 140: 7-8 System Maintenance To replace t
Page 141 and 142: 7-10 System Maintenance To replace
Page 143 and 144: 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
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Materials Required The following ma
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Extracting Pure Dye Information fro
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Constructing a Custom Pure Dye Plat
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Verifying Instrument Performance Us
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Preparing and Running an RNase P Pl
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Adjusting the Sensitivity of the Pl
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Testing the Adjustment 7-30 System
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Aligning the Plate Handler When to
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7-34 System Maintenance To align th
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Re-checking the Input Stack 1 7-36
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Defining the Positions of the Remai
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Aligning the Fixed-Position Bar Cod
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7-42 System Maintenance To position
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7-44 System Maintenance
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General Computer Maintenance Mainte
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Maintaining the SDS software Admini
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Troubleshooting Table 8-2 Troublesh
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Low Precision or Irreproducibility
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Writing on the Reaction Plates Fluo
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Background Runs Background Troubles
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Pure Dye Runs Pure Dye Troubleshoot
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8-12 Troubleshooting Troubleshootin
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Software and 7900HT Instrument Trou
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8-16 Troubleshooting Troubleshootin
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User Bulletins 9 9 About This Chapt
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Fluorescent-Based Chemistries Funda
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Fluorescence Detection and Data Col
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Normalization of Reporter Signals A
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Determining Initial Template Concen
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Calculating Threshold Cycles A-10 T
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Importing and Exporting Plate Docum
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Configuring the Setup Table File wi
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About the Setup Table File Format S
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Setup Table Elements (continued) Nu
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Exporting Plate Document Data Expor
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Designing TaqMan Assays C In This A
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Design Probes and Primers The follo
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Design Tips for Allelic Discriminat
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Kits, Reagents and Consumables D In
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Consumables and Disposables The ABI
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TaqMan Pre-Developed Assays and Rea
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Contacting Technical Support F Serv
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G-2 Limited Warranty Statement No a
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A(continued) Automation Controller
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I(continued) installing plate adapt
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Q quantifying probes and primers C-
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Headquarters 850 Lincoln Centre Dri
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