Endpoint Genotyping This technical note describes how to set up, perform and analyze genotyping assays (allelic discrimination assays) to detect known single nucleotide poly- morphisms (SNPs) using the LightCycler® 1536 Real-Time PCR System and the LightCycler® 1536 Endpoint Genotyping Analysis Tool. Dual color hydrolysis probe assays Each hydrolysis probe contains two labels, a fluorescent reporter and a quencher, in close proximity to each other. In addition to the target specific primers, endpoint genotyping analysis uses two sequence-specific probes that are designed to detect allele X and allele Y, respectively and are labeled with different reporter dyes. Hydrolysis probes specificity: J FAM dye detects samples that are homozygous for allele X and heterozygous XY. J VIC (HEX) dye detects samples that are homozygous for allele Y and heterozygous XY. A fluorescence signal is generated when dyes, bound to allele-specific probes, are cleaved. The intensity of the signal depends on the amount of hybridization between probe and target, which will be greater for a perfect sequence match than for a mismatch. The endpoint fluorescent signal can be measured by the LightCycler® 1536 Instrument. LightCycler® 1536 Software automatically calculates endpoint fluorescence (EPF) values after the PCR run is finished. EPF values can be transferred, via export/ import commands, from the LightCycler® 1536 Software into the LightCycler® Endpoint Genotyping Analysis Tool. This tool can be used to group samples based on their endpoint fluorescence intensity and to classify these groups according to genotype (homozygous X, homozygous Y, heterozygous XY). Results can be displayed as a scatter plot, result table or plate view. Topic Purpose of this Technical Note Assay Principle LightCycler ® 1536 Real-Time PCR System LightCycler® 1536 Endpoint Genotyping Analysis Tool Technical Note Detection Format Assay Results
Transcript
Endpoint Genotyping
This technical note describes how to set up, perform and analyze genotyping assays (allelic discrimination assays) to detect known single nucleotide poly-morphisms (SNPs) using the LightCycler® 1536 Real-Time PCR System and the LightCycler® 1536 Endpoint Genotyping Analysis Tool.
Dual color hydrolysis probe assays Each hydrolysis probe contains two labels, a fluorescent reporter and a quencher, in close proximity to each other. In addition to the target specific primers, endpoint genotyping analysis uses two sequence-specific probes that are designed to detect allele X and allele Y, respectively and are labeled with different reporter dyes.
Hydrolysis probes specificity:J FAM dye detects samples that are homozygous for allele X and heterozygous XY. J VIC (HEX) dye detects samples that are homozygous for allele Y and heterozygous XY.
A fluorescence signal is generated when dyes, bound to allele-specific probes, are cleaved. The intensity of the signal depends on the amount of hybridization between probe and target, which will be greater for a perfect sequence match than for a mismatch. The endpoint fluorescent signal can be measured by the LightCycler® 1536 Instrument.
LightCycler® 1536 Software automatically calculates endpoint fluorescence (EPF) values after the PCR run is finished. EPF values can be transferred, via export/ import commands, from the LightCycler® 1536 Software into the LightCycler® Endpoint Genotyping Analysis Tool. This tool can be used to group samples based on their endpoint fluorescence intensity and to classify these groups according to genotype (homozygous X, homozygous Y, heterozygous XY). Results can be displayed as a scatter plot, result table or plate view.
RealTime ready DNA Probes Master 05 502 381 0015x conc., 5 x 1 ml (12,500 x 2 µl reactions)
Roche Applied Science - Ordering Information
Products from Roche Applied Science: 1) For detailed information using the LightCycler® 1536 System, please refer to the LightCycler® 1536 Operator’s Guide.
2) For further details on the LightCycler® 1536 Endpoint Genotyping Analysis Tool, please contact your local Roche representative.
3) For detailed information using the RealTime ready DNA Probes Master, please refer to the package insert for this product.
For detailed information regarding the LightCycler® 1536 System, please visit: www.lightcycler1536.com.
Other products which were used for the setup of the LightCycler® 1536 Multiwell Plate in the sample experiment described in this Technical Note:
4) Sealing foil: Clear Weld Seal Mark II, 4titude®, Ltd., UK
5) Semi-automated heat sealing: Agilent PlateLoc Thermal Microplate Sealer, Agilent Technologies, CA, USA
6) Low-volume dispenser used for this example: InnovadyneTM NanodropTM Express (1x 16 head), IDEX Health & Science LLC., WA, USA
The workflow times listed in this example refer to an experiment that analyzes 16 SNPs for 96 different genomic DNA samples. The calculated total
time (~ 155 minutes) is valid only for that particular plate layout and workflow, as performed with the equipment listed above.
I. Manual preparation of Source Plates (96-well format):
Source Plate I: PCR reaction mix (RealTime ready DNA Probes Master, primers, probes) Source Plate II: Genomic DNA samples (with optional addition of Setup Control)
II. Automated preparation of Destination Plate (LightCycler® 1536 Multiwell Plate): Automated dispensing of equal volumes of PCR reaction mix (from Source Plate I, 1 µl) and DNA samples (from Source Plate II, 1 µl) into each destination plate well, using a low-volume liquid dispenser.
III. Processing of the loaded Destination Plate (LightCycler® 1536 Multiwell Plate): - Heat sealing - Centrifugation
IV. Performing the LightCycler® 1536 Real-Time PCR run (45 cycles)
V. Exporting the LightCycler® 1536 result table (*.txt) from the LightCycler® 1536 Software to a specified directory
VI. Importing the *.txt file into the LightCycler® 1536 Endpoint Genotyping Analysis Tool
VII. Performing a manual endpoint genotyping analysis within the LightCycler® 1536 Endpoint Genotyping Analysis Tool
VIII. Optional: Exporting the results of the endpoint genotyping analysis to a dedicated folder in a specified directory for storage
~ 20
~ 60
~ 5
Time(min.)
~ 15
~ 50
~ 5
1536 Endpoint Genotyping
Semi-A
utomated P
CR
Plate-SetupP
CR
Run / A
nalysisD
ownstream
Analysis
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Different source plates must be prepared, depending on the specific automatedlaboratory workflow used, to set up the LightCycler® 1536 Multiwell Plate (Destination Plate).
In the example described below, two source plates (96-well format) are used forthe final setup of the LightCycler® 1536 Multiwell Plate:
1. Semi-Automated Setup of the PCR Plate
Figure 2: Setup Scheme: Sample experiment for endpoint genotyping analysis of 16 SNPs for 96 genomic DNA samples,
including no template controls (NTCs).
Preparation of Source Plates Preparation of Destination Plate
JSource Plates can be filled manually or set up using automation.
JTo simplify the experimental workflow, in most routine ex- periments, you can pre-plate either the genomic DNA samples or SNP Genotyping Assays (whichever of these components has the higher complexity), and dry down that component on the plate.
JAutomated setup of the Destination Plate using a low-volume dispenser.
Destination Plate:
(1536-well format)
2 µl/well
(1 µl)
(1 µl)
(96-well format)
96 samples
Source Plate II:
JGenomic DNA samples
JSetup Control (optional)
Source Plate I:
JRealTime ready DNA Probes Master
JSNP Genotyping Assays (primers, probes)
JWater, PCR Grade
16 SNPs
(96-well format)
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J Pipette the appropriate volumes of the different components into Source Plate I.
Source Plate II: Genomic DNA (optional with Setup Control)
J Prepare 2x concentrated aliquots of the genomic sample DNAs. The final DNA concentration in the aliquots should be 2 to 10 ng / 1 µl. J Pipette the 2x concentrated sample DNAs and an aliquot of the 20x concentrated Setup Control into Source Plate II:
Automated Dispensing of the Destination Plate and Plate Processing:
J Dispense equal volumes (1 µl) of the PCR reaction mix (Source Plate I) and DNA samples (Source Plate II) into wells of the Destination Plate (LightCycler® 1536 Multiwell Plate) using a low-volume liquid dispenser.J Heat seal the multiwell plate with adequate sealing film.J Place multiwell plate in a standard swing-bucket centrifuge and centrifuge for 2 minutes at 1,500 x g.J Load the multiwell plate into the LightCycler® 1536 Instrument.
J Calculate the total volume of a 2x concentrated PCR reaction mixture containing the 5x Master Mix and the specific PCR primers and probes.
J Prepare a 20x conc. SNP Genotyping Assay Mix that contains all specific PCR primers (18 µM each) and both hydrolysis probes (FAM-labeled and VIC (HEX)-labeled, 5 µM each).
ComponentsVolume (µl)for One PCR Reaction (2 µl)
Sample experiment: Volume (µl) for 96 PCR Reactions*
RealTime ready DNA Probes Master,Master Mix, 5x conc.
NOTE* Sample experiment: Endpoint genotyping analysis for 96 genomic DNA samples per SNP.
** The total required volume of each reagent must include sufficient extra volume to offset (a) reagent pipetting losses and (b) other losses (e.g., dead volume) that depend on the specific low-volume liquid dispenser used to transfer reaction mix to the Destination Plate.
NOTE* For the subsequent target-specific endpoint genotyping analysis to operate properly, each item in the sample list must contain both a sample name and a target name.
To program a new experiment for endpoint genotyping analysis:
J Create a new experiment.J Set the detection format: Dual Color Hydrolysis Probes / UPL Probes.J Define programs: Preincubation, 2 Step Amplification, Cooling (Keep default parameters for all programs).J Select the pipetting control mode according to your chosen experimental setup: Setup Control or Master Control.
J Import a sample list containing sample names and target names*.J Add notes regarding the experiment (optional). J Start the PCR run on the LightCycler® 1536 Instrument.J Save the experiment. J At the end of a successful run, the results are automatically displayed in two formats (result table, amplification charts).J Export and save the Result Table.txt file from the run to a dedicated folder.
3. LightCycler® 1536 Endpoint Genotyping Analysis Analysis of EPF Values using the LightCycler® 1536 Endpoint Genotyping Analysis Tool
NOTERequirements for using the LightCycler® 1536 Endpoint Genotyping Analysis Tool:J The tool is an Excel template (.xlt file) that requires Microsoft® Excel 2003 or later version. J LightCycler® 1536 Software (version 1.0) expresses floating point numbers in the English (USA) format. Therefore you must use the English version of Microsoft Excel and set the Microsoft Windows Regional and Language Options to English (USA).J Recommended screen resolution for the computer monitor: 1236 x 1024.
To analyze the EPF values generated with the LightCycler® 1536 Instrument / Software:
J Open the LightCycler® 1536 Endpoint Genotyping Analysis Tool (LightCycler® 1536 Endpoint Genotyping Analysis Tool.xlt file) in Microsoft Excel.
J If using Microsoft Excel, version 2003: select Activate macros in the Excel wizard that appears or J If using Microsoft Excel, version 2007: choose Options under “Global Selection”, then select Security Warning - Macro and check Enable this content.
NOTEThe Excel Analysis sheet is protected: Sorting and writing is disabled, filtering is enabled.
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A) Symmetric Auto-Scaling
(both axes have the same scaling).
B) Axis Specific Auto-Scaling
(scale of each axis was set according
to the maximum fluorescence value
seen in each channel).
A B
Figure 3: Description of the Analysis Screen and its adjustable functions.
Angles for FAM and VIC Zone, respectively:Determines areas of the scatter plot within which samples are called Homozygote FAM or Homozygote VIC. The size of the area in which samples are called Homozygous for the allele that binds the FAM or VIC probe can be manually adjusted using the corresponding up/down buttons. Threshold Pos/Neg: Determines the area of the plot within which samples are called Negative. The size of the area in which samples are called Negative can be manually adjusted using the corresponding up/down size tabs. Angle Unknown Zone: Determines the area of the plot within which samples are called Unknown. The size of the area that defines samples of Unknown genotype can be manually optimized (using the corresponding up/down size tabs) to meet specific experimental requirements.
1 2
3
4
Spin buttons Scatter plot Filter function
Sample list
Negative
Detector
Unknown
Detector
Homozygote
Unknown
Homozygote
Drop-down-buttonfor target selection
Heterozygote
1
2
3
4
Display options for the scatter plot (drop-down menu, upper right corner of screen):
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J If multiple genotype targets are analyzed in a single LightCycler® 1536 Instrument run, as in the sample experiment described here, you must manually perform the following analysis steps for each geno- type target (e.g., ADD1, TGF beta):
J Select the first genotype target via the drop-down tab in the Target column.
J Using the relevant spin buttons, adjust the settings for Angle FAM Zone, Angle VIC Zone, Angle of Unknown Zone, and Threshold Pos/Neg.
J Click the “Submit to Result Tab” button to copy the calculated genotypes of the currently selected target into the result table (Excel Result sheet).
J Repeat the above three steps for each genotype target in the experiment.
J To view the genotyping calls for all targets, select the Result tab. Genotypes displayed on the result table (Excel Result sheet) are based on the individual angle and threshold settings that were manually selected for each target.
NOTEThe Excel Result sheet is not protected; both read and write functions are enabled.
Figure 3: Description of the Analysis Screen and its adjustable functions.
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J Click the “Plate View” button to display the genotype calls arranged on a schematic of the LightCycler® 1536 Multiwell Plate. If desired, click the “Print” button, and the plate view will be printed to the default printer.
J Click the “Export Result.txt” button to save the data displayed on the Excel Result sheet as a .txt file.
J In both versions, Microsoft Excel 2003 and Microsoft Excel 2007, save the entire file (i.e., both the Analysis and Result sheets) as an Excel 97-2003 workbook (*.xls).
NOTEA complete overview of all the target-specific results obtained for the analyzed samples is shown only on the Excel Result sheet; the data displayed on the Excel Analysis sheet is only for one genotype target at a time.
NOTEWhen working with Microsoft Excel 2007, do not save the file as an Excel 2007 workbook with macros (*.xlsm) or as an Excel 2007 template with macros (*.xltm). When the file is saved in either of these formats, its macros may be permanently disabled, especially if no antivirus protection is installed on your PC.
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