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Manual - VTP UP

1. Chapter 4 The HR 1 Instrument Control Software 0 HR 1 Instrument Serial Port Selection Select the correct Serial Port from the File menu The information bar at the bottom of the software window will give information about the HR 1 instrument communication with the Tea PC The information bar will display 6 connection status PC Com port machine ID current user melting pro tocol being used and the status of the machine melting holding cooling etc Select the proper Com port that is attached to the HR 1 instrument View Menu Under the View menu there are two options for display status The Real Time Graph will display a split screen where the user can observe in real time the temperature of the heated ingot and the continuous fluores cent status of the sample in the top screen The corresponding melting curve data fluorescence vs temperature will appear in the lower screen The Melting Graph option will display only the melting curve of the sample the lower screen from the Real Time Graph option in the full screen of the software window but will not display the temperature or fluorescence over time A digital display of the temperature and fluorescent signal is always present in the information bar just below the menu bar at the top of the soft ware window Pause Data and Clear Data are also options in this window refer to definitions at the beginning of this chapter HR 1 I
2. 1 Unpack the instrument by carefully removing it from the sealed box Refer to the packing list to ensure that all of the components are included 2 Place the instrument on a clean well ventilated lab bench Make sure there is 10 cm of space behind the instrument to ensure adequate airflow for cooling of electronic components Transport the instrument by placing one hand underneath and one hand on top or on the side of the instrument Do not carry the instrument by the hinged lid HR 1 Instrument Placing the instrument in a room that is kept at a relatively cool temperature A will facilitate more efficient cooling of the heated ingot upon completion of a melt 20 25 C 3 Connect the USB to serial converter to the laptop and then connect the serial cable between the converter and the HR 1 s serial port It is not recommended to connect or disconnect the USB serial cables while the laptop is running 4 Make sure the power switch located on the back of the instrument is in the OFF position before you plug in the power cord Connect the HR 1 and laptop power cord to a grounded power supply 5 The instrument is now ready to be turned on Serial Port Power Switch Power Cord receptor HR 1 Instrument 19 Instrument Specifications and Installation Chapter 2 Software Installation The HR 1 Software Package is designed specifically for Microsoft Windows 2000 or XP Operating Systems Administra
3. 2 Replace with properly rated fuses Insert assembly into mounting and snap it in place Fuse Access Serial Plate HR 1 Instrument 57 Troubleshooting Chapter 6 Software LED Seek Warnings or Failures Solutions 1 Re melt sample 2 The sample is not giving enough fluorescence to reach the required levels specified in the melting protocol Adjust target fluorescence percentage or re optimize amplification protocol to obtain higher fluo rescence Auto Set Fluorescence is used all samples should be analyzed using the same target fluorescence level Chapter 6_ Troubleshooting 8 HR 1 Instrument HR 1 Instrument 59 Return amp Decontamination Forms Appendix A Appendix Return amp Decontamination Forms To return a machine or product for replacement or repair follow the basic guidelines for decontaminating and returning the machine or product Once the forms have been filled out and the appropriate steps have been taken fax or e mail the forms to Idaho Technology at 801 588 0507 or it idahotech com The following forms have been provided Equipment Return Procedure e RMA Return Fax e Return amp Repair Form Decontamination Labels For more information please visit our Web site at www idahotech com or by phone 1 800 735 6544 or 1 801 736 6354 Appendix A_ Return 8 Decontamination Forms 0 HR 1 Instrument Equipment Return Procedure Ob
4. e DO a 10 Fluorescence F1 Temperature C Temperature C The Derivative Plot and the Difference Plot of two heterozygous samples with different mutations from previous figures Note the differences between the two heterozygous samples HR 1 Instrument 53 Troubleshooting Chapter 6 CHAPTER Troubleshooting Introduction This chapter details some problems that may arise with the instrument or software and steps you can take to resolve these issues It is a simple trouble shooting guide If a problem persists with the instrument or soft ware please contact Idaho Technology s customer support at 1 800 735 6544 Hardware Before attempting any manipulation of the hardware described in this chap ter turn off and unplug the instrument from any power source and discon nect the serial cable from the computer Cleaning the Ingot after Breaking a Capillary A Before attempting to clean the heated ingot cool the ingot to a temperature that is safe to handle 40 C or below Wear gloves before attempting to clean the ingot with a broken capillary tube Change gloves often during the procedure to reduce the risk of con tamination i Troubleshooting 4 HR 1 Instrument Solution Turn off the instrument and unplug it from the electrical outlet before begin ning the procedure for cleaning the ingot supplied in the HR 1 Tool Kit to remove the two small screws that hold the plastic co
5. H I GH HR 1 RESOLUTION reer he May Sy NN Ng scanning Re EONI Gere p gt Ol dif AAA 1 fe fete Bt ie 6 Technical Users Manual Status Indicators Lid Catch Baffle HR 1 Instrument 1 Introduction 2003 Idaho Technology Inc All rights reserved Part HR01 PRT 0008 HR 1 Technical Users Manual Rev 01 Information in this document is subject to change without notice No part of this document may be reproduced or transmitted in any form or by any means electronic or mechanical for any purpose without the express written permission of Idaho Technology Inc HR 1 Instrument Control software HR 1 Data Analysis software modules 2003 Idaho Technology Inc RapidCycler and R A P 1 D are registered trademarks and LCGreen HR 1 are trademarks of Idaho Technology Inc LightCycler is a trademark owned by a member of the Roche group U S Patent Number 6 174 670 foreign counterparts and pending patents This product is for research use only Printed in the United States of America Idaho Technology makes no warranty of any kind with regard to this material including but not limited to the implied warranties of merchantability and fitness for a particular purpose Idaho Technology shall not be liable for errors contained herein or for incidental consequential damages in connection with the furnishing performance or use of this material Safety and Regulato
6. Ele a Final Results Sample 1 Sample 2 5 Exonz Fragment H Condition 1 H E Condition 2 H 3 0 Final Results B Target Name Gene 2 Exon 1 Fragment H Condition 1 H Condition 2 i a Final Results 63 Exon Fragment H Condition 1 H Condition 2 9 Final Results to compare all at once The analysis software will automatically display the differences in the pathways to the samples in the sample names column allowing easy identification of which samples you are analyzing and what the different experimental conditions were for each sample For example to analyze Sample 1 and 2 from both experimental conditions in Exon 1 the sample name list would look like Condition 1 Sample 1 Condition 1 Sample 2 Condition 2 Sample 1 Condition 2 Sample 2 Once the optimal experimental and melting conditions have been deter mined a Final Results folder can be created to perform the definitive analy sis on all samples allowing easy anal ysis for all samples under the same final conditions and keeping data well organized in a standard format Melting a Sample After the capillary is properly seated in the ingot Chapter 2 loading and unloading a sample close the lid Chapter 4 _ The HR 1 Instrument Control Software _ 8 HR 1 Instrument and select Start Melting in the Melting menu Use the pull down menu to select the protocol Select Edit Pro
7. To display the Derivative Plot choose Derivative Plot under the Analyze menu The negative derivative of fluorescence is plotted on the Y axis with Temperature on the X axis Melting peaks are displayed allowing easy visualization of differences between the samples The peak of the curves is known as the Tm Melting Temperature Tm is the temperature at which the DNA duplexes are denaturing at their greatest rate The Derivative Plot may be useful for identifying differences in the type of mutation present in individual samples The Derivative Plot analysis option is the only analysis option available if the melting curves have not been normalized Cutting and Pasting Graphics To cut graphics from the analysis ss alan software hold the CTRL key while simultaneously selecting the PRINT SCRN key on the computer keyboard CTRL Print Scrn Paste the image into an appropriate graphics handling program as a Bitmap BMP using the CTRL V function Y is E o a 5 5 8 6 4 2 o 2 44 8 88 89 90 a1 Temperature Final Note on Analysis Software There is much data analysis functionality within the software that can be found by experimentation The instructions provided in this User s Manual are sufficient for familiarization required to perform practical and functional analysis of data and give the basics for further analysis Chapter 5_ HR 1 Analysis Software 2 HR 1 Instrument
8. Adobe Acrobat PDF for mat by selecting the User Manual option under the Help menu Selecting the About HR 1 menu selection will bring up the about win dow describing the software version There are two selectable options OK and Reset Selecting OK will return to the main screen Selecting Reset performs the same function as the Reset Instrument selection in the Tools menu If the problem is not resolved with the instrument or software please call Idaho Technology s customer service Data Organization It is important to organize the data you will collect with the HR 1 instrument Setting up a main folder for all of the data that will be collected is recom mended with separate folders within the main folder that identify different targets experimental conditions users etc All fluorescence and tempera ture data for each sample is stored in a single file vs2 extension Based on the software characteristics an example is included of how to organize and name different folders and files for efficient data retrieval Chapter 4 i The HR 1 Instrument Control Software HR 1 Instrument This method of data organization will allow the selection of individual sam ples for analysis or multiple samples from different experimental conditions Ela all HR 1 Data 3 Target Name Gene 1 5 a Exon 1 Fragment 2 Condition 1 Sample 1 Sample 2 Condition 2 Sample 1 Sample z
9. Display options ceeeeeeeeees Diference Derivative Plot 2 cccccscccceesseeceeeeeseeceteessceeenenesseneeees Cutting and Pasting Graphics ecceeeeeeeeeeeeeeenees Final Note on Analysis Software n e Chapter 6 Troubleshooting Hardware Cleaning the Ingot if a Capillary 376868 Lid will not unlock after run Instrument does not respond to computer prompts Instrument does not respond when power is turned on Software LED Seek Warnings or Failures Appendix A Equipment Return Procedure cccccceeeeeeeteeeeeennteeees Return Materials Authorization Return amp Decontamination Labels 000 cece eee HR 1 Instrument 5 Introduction Introduction Idaho Technology the creators of melting curve analysis introduces the new powerful and affordable platform for post amplification genotyping gene scanning and SNP mutation detection Utilizing a closed tube DNA amplification reaction with our new LCGreen I Dye and the HR 1 High Resolution Melter users are able to detect different DNA species by observ ing subtle differences in fluorescent signal over temperature changes The system can reduce sequencing efforts and cost by greater than 90 by bringing scanning efforts to the bench The HR 1 System utilizes Roche LightCycler glass capillary tubes in an aluminum thermal cylinder using si
10. The raw data from each sample selected will be displayed on the initial screen with the pathway to the samples shown as the Graph Title and indi vidual Sample Names displayed on the selectable upper left portion of the graph To open additional or new sample files select Add Files or New Files in the File menu respectively Add Files automatically returns to the last directory selected Selecting New Files opens the default browser window HR 1 Instrument 47 HR 1 Analysis Software Chapter 5 Normalizing Data The first step in analyzing the sam pen ples is to normalize the fluorescence 0 data by selecting Normalize inthe Spez Analyze menu This opens a window that shows 4 vertical cursors num bered 1 4 Cursors 1 and 2 should be moved select and hold with mouse to move to identify a linear region of the melt ing curves prior to the major melting transition of the sample s Cursors 3 and 4 should be moved to identify a linear region of the melting curve s following the melting transition of the sample s The cursors must be kept in the same numeric order relative to each other from left to right When normalizing the melting curves the original data will appear in the upper panel of the screen while the lower panel of the screen will show a preview of the final curves after normalization The cursors are initially placed at default positions although it is often necessary to adjus
11. base paired molecules In the past distinguishing heteroduplex DNA molecules from fully base paired molecules using fluorescent melting curve analysis met with lim ited success Two factors contributed to the inability to effectively detect heteroduplex molecules 1 Inadequate thermal control of the instrumenta tion and 2 shortcomings of dsDNA binding dyes With the development of LCGreen and the improved temperature control and resolution of the HR 1 instrument these issues are resolved in a single cost effective and simple to use platform HR 1 Instrument 9 HR 1 Scanning Principles Chapter 1 Wildtype Bos 105 Hetero 100 v 9 jA o 9 a 5 2 i 82 83 84 Temperature Temperature The left panel shows the melting curves of a wild type dark line and a heterozygous light line sample Note the lower temperature inflection point in the heterozygous sample relative to the fully base paired wild type sample The right panel shows the derivative plots from the melting curves Note the single peak in the curve generated by the wild type specimen as compared to the dual phase peak generated by the heterozygous sample Resolution and Reproducibility Two samples one wild type and one heterozygote were amplified Analysis was completed and repeated 30 times on HR 1 to measure intra sample variability Sample temperatures were recorded at 5 increments of nor malized fluorescence for each of the 3
12. contact for a minimum of 10 minutes HR 1 Instrument 61 Return amp Decontamination Forms Appendix A Decontamination Labels After the above steps have been completed the person responsible for the return must complete and sign two Decontamination Labels and attach one to the instrument and the other to the exterior of the shipping container Complete and sign the Decontamination Form a photocopy should be made for your records and the original must be returned with the instrument A Return Materials Authorization Number RMA must be obtained from Idaho Technology prior to shipment and the RMA and Decontamination Labels must be visible on the exterior of the shipping container Idaho Technology reserves the right to return and or refuse receipt of any materials at the customer s expense that do not meet these requirements Packaging and Shipping The HR 1 case is to be wrapped in a plastic bag before it is inserted into the box There is to be at least 2 inches of foam or packaging material on the bottom of the heavy weight box recommended box size 24 x 20 x 12 Place the wrapped HR 1 back side down with the start up kit directly next to the machine Place 2 or more inches of foam or packaging material all around the instrument International Shipping If a HR 1 instrument is shipping internationally it MUST have a license and is to be shipped via an Idaho Technology preferred carrier To find
13. 0 independently assayed samples Sample temperature standard deviations ranged from 0 006 0 045 C at normalized fluorescence of 10 and 90 respectively In addition 17 dif ferent wild type and ten different heterozygote samples were amplified and analyzed for a measure of inter sample temperature variance Standard deviations were of similar values with a slightly tighter range data not shown These results demonstrate the temperature homogeneity both within and between samples analyzed with LCGreen HR 1 Increased Resolution with LCGreen 1 dsDNA binding dye In previous attempts at sequence variant scanning by fluorescent melting curve analysis the primary shortcoming of dsDNA dyes was a strong inhibi tory effect upon amplification at dye concentrations required to sufficiently saturate the newly synthesized product A consequence of using dsDNA Chapter 1_ HR 1 Scanning Principles _ 0 HR 1 Instrument binding dyes below a saturating concentration is that the dye molecules redistribute from regions undergoing thermal denaturation to regions that remain double stranded Dye redistribution effectively eliminates the abil ity to detect the subtle fluorescence differences characteristic of heterodu plexes via thermal denaturation LCGreen can be used at concentrations high enough to saturate the available double stranded binding sites without inhibiting amplification This characteristic assures product saturati
14. 801 736 6354 International Reach Us By Fax To contact Idaho Technology by fax use the following numbers 1 801 588 0507 United States and Canada IDD Prefix 1 801 588 0507 International HR 1 Instrument 3 Introduction Table of Contents ay Caco 0 3 1 Maru Re ee 5 Chapter 1 Principles of Mutation Scanning and Genotyping ai e EE AAE E E A A AAN O E A E N EE fetadedass 7 Heteroduplex detection snuro aren e e ai anaiari 7 Resolution and Reproducibility 9 Increased Resolution with LCGreen dsDNA binding dye ceeeeee 9 Genotyping with HR 1 and LCGreen o oo eeceeeeeeeeeeneeeeeeeeeeeeeseeeneeeers 10 Product Size and Domain Melting cssesinsrisriiiniiiiia 10 Chapter 2 Instrument Specifications and Installation 13 UG ON 00 The AR 1 Instr ment 5 15 Reagent REcommendations ccccccceeceeeeeeeeeeeeeeeeeeeenneeeeeeenieeeeeseneeeeeree 16 FIR 1 SPOCiiCatlONS es cist ccedseswsdstsscsastincodessd 0 17 17 ea E AN aY 19 300 Software Chapter 3 HR 1 General Operating Instructions 21 0 0 000 General Considerations ccccccccccceeeeeeeceeeeeeeeeeeeeeeeeeeeeecceeeeeeeeeeeeeeeteeeeees 21 Preparation of a DNA Sample cccceeeeeeeeeeeeeeecnneeeeeeeeeeeeeeeeeenennaeees 22 aaaea aaae aaia aE 22 Loading and Unloading a Sample ccccceeeeeeeeeeennee
15. It is not neces sary to add this to the sample if the sample already contains buffer with BSA Buffered MgCl2 solutions for amplification is to be used at 1 10 con centration supplied as 10X includes BSA Enzyme diluent is to be used to dilute enzyme includes BSA HR 1 Instrument 17 Instrument Specifications and Installation Chapter 2 HR 1 Specifications Temperature sensing is acquired using a platinum resistive thermal device RTD embedded inside the machined cylinder Fluorescence detection is initiated with an excitation source that is compensated for temperature drift and stability Intensity of the excitation source is controlled by a 12 bit digital to analog converter that varies the current to the excitation source Fluorescence detection is achieved using a single wavelength silicon based hybrid photodiode and amplifier Ramp rates of 0 01 C to 1 0 C are attainable from ambient temperature to 100 C Control of the HR 1 instru ment is serial based to a laptop computer Instrument requires 100 240 VAC 50 60 HZ and consumes approxi mately 40 Watts of power 12W max single sample heater with 12 bit control 28mA max LED excitation with 12 bit control 24 bit temperature and fluorescence detection Optics include single color excitation and single color detection Temperature ramp rates from 01 to 1 C per second Temperature ramp range from ambient to 100 C Setting Up the HR 1 Instrument
16. Signature Date User must contact Idaho Technology prior to returning the instrument to receive an RMA HR 1 Instrument 63 Return amp Decontamination Forms Appendix A Return Materials Authorization Fax pg 2 of 2 If your machine is still under warranty please supply the purchase date and machine serial number if your machine is out of warranty please supply a blank PO for the repair and shipping charg es Please note that your item must be returned with an Idaho Technology assigned RMA and Decontamination documentation Prior to returning the item you must contact Idaho Technology for an RMA number and also fax copies of all necessary Decontamination Forms to Fax 801 588 0507 Attn Technical Support All returns should be sent to the following address Idaho Technology Inc 390 Wakara Way Salt Lake City UT 84108 USA 801 736 6354 Declaration of Decontamination This instrument has be decontaminated in accordance with an established government regulation or equivalent rendering it free from radioactive biological or chemical contamination Which Method was Used What chemical infectious toxic or radioactive substances have been in contact with this product Also indicate if flammable or corrosive Authorization Notice By accepting this authorization to return this product the user assumes all responsibility for decon tamination and cleaning Idaho Technology Inc reserves the right to ref
17. a pre ferred carrier for your region contact our shipping department at 801 736 6354 A Return Materials Authorization Number RMA must be obtained from Idaho Technology prior to shipment and the RMA and Decontamination Labels must be visible on the exterior of the shipping container Idaho Technology reserves the right to return and or refuse receipt of any materials at the customer s expense that do not meet these requirements Appendix A_ Return 8 Decontamination Forms 2 HR 1 Instrument Return Materials Authorization Fax pg 1 of 2 From Pages Attn Ship to Idaho Technology Inc Warranty 390 Wakara Way Non Warranty Salt Lake City UT 84108 USA 801 736 6354 Date RMA Return Materials Authorization Company Department PO Phone Fax Model Serial No Notes Please read and completely fill out the Return Machine Packet found on the Internet at http www idahotech com product_sup forms htm The decontamination form and labels must be completed before the machine is returned to Idaho Technology In the event the unit is returned without documentation it will be shipped back to you at your expense Non warranty charges are 70 hr with a 2 hr minimum charge plus parts and shipping charges In the event the unit cannot be repaired it will either be returned to you or disposed of upon your request Thank you for your business and support of the health and well being of our employees
18. cally be shown Wie r as in the following image ji The heterozygous site may ee r involve one or more nucleo Allele 2 Mutant G tide substitutions insertions e area or deletions If these prod c ucts are heat denatured and Chapter 1_ HR 1 Scanning Principles 8 HR 1 Instrument then allowed to re anneal four DNA duplex species are formed The double strand molecules with non Watson Crick base pairing mismatches are described as heteroduplexes The perfectly matched duplexes are consid ered fully base paired Mismatch base pairing results ies Created in heteroduplex molecules that have unique physical 2 Homeduplexes amp properties The differing eo physical properties of hetero duplexes include the tempera gt Heteroduplexess 7 7 ture at which the strands dis associate or melt Differing physical properties between heteroduplex molecules and those having fully complimentary base pair ing is the basis by which the different species are distinguished to identify heterozygous polymorphisms or mutations During slow controlled heating of a heteroduplex mismatch base pairing causes the strands to disassoci ate at a lower temperature than fully base paired molecules LC Green I a fluorescent double stranded DNA dsDNA binding dye facilitates the obser vation of this melting process seen as an early loss of fluorescence com pared to a sample containing only fully
19. dure it is possible to collect the relevant data within a smaller temperature range minimizing the amount of time required per sam ple HR 1 Instrument Panel B 40 Fie eit Seis Analyse Deploy Mote Stye About Tm 83 8 C Set melting protocol parameters for acquisition at 79 C with final at 87 C Temperature Samples test melt 1 2 7 32 Chapter 4 The HR 1 Instrument Control Software Fle Est selen Analyze Dipy Pat Siye About Samples test melt 1 5 Fluorescence E E E AE E E E ie She 90 89 88 87 86 85 84 83 82 81 80 79 78 77 76 75 74 73 72 74 70 Temperature Use caution in setting the lower temperature as some heteroduplex transi tions can occur 5 10 C below the primary melting transition HR 1 Instrument 41 HR 1 Analysis Software Chapter 5 CHAPTER HR 1 Instrument Analysis Software Introduction This chapter describes the basic concepts about the HR 1 Data Analysis software package Chapter 5 is a step by step guide to the analysis pro cess HR 1 Melt Analysis Tool Analysis Software Overview Menu Bar File New Files Import new vs2 files for analysis Add Files Add vs2 files to current analysis Sample Names As Input default setting displays sample names as they Chapter 5_ HR 1 Analysis Software 2 HR 1 Instrument were originally entered Unique Characters displays only the un
20. e Derivative peaks give an approximation of the melting temperature Tm of a sample Display Show Cursors Displays vertical and horizontal adjustable cursors Show Run Data Displays information about the melting parameters that were used on the samples Show Graph Tools Displays some basic graph tools on the chart allowing the user to manipulate the appearance of the graph Graph Title File Name Default setting displays the graph title as the pathway to the files that are being analyzed Rename Allows the alteration of the name of the graph Show SIN Displays the Signal to Noise ratio for each sample in a tabular format Plots All Default setting displays the melting curves of all the samples Chapter 5_ HR 1 Analysis Software _ 44 HR 1 Instrument None Clears the display area of all melting curves Every 2nd displays every 2nd sample 3rd displays every 3rd sample 4th displays every 4th sample Subset 1st half displays the first half of the sample list 2nd half displays the second half of the sample list 1st third displays the first third of the sample list 2nd third displays the second third of the sample list 3rd third displays the last third of the sample list Invert Clears the samples that are currently displayed and displays all other samples on the list Style Color Default setting sample curves are displayed in different colors with various line and point st
21. ed for this to operate New Protocol Opens the Edit Melting Protocol dialogue window Allows the user to create a new protocol Edit Protocol Opens the edit Melting Protocol Dialogue window HR 1 Instrument 27 The HR 1 Instrument Control Software Chapter 4 Allows the user to view and edit an existing protocols Cancel Closes Start Melting dialog window Melting Analysis Launches the HR 1 analysis software if installed Abort Melting Stops the current melting experiment Existing data is saved under the experiment name It will also abort a hold Melting Protocols Opens melting protocol directory Protocols can be examined and edited right mouse click LED Accesses the LED controller options Adjust LED Power Accesses the LED power dialog window Power Slide Bar Use to manually adjust LED power setting Power output will be indicated by percentage in the dialog window Close Exits Adjust LED Power dialog window Save Saves adjustments made to the LED power level to the current melting protocol Turn LED on Turns LED on when not in an active melting protocol Turn LED off Turns LED off when not in an active melting protocol Tools Accesses HR 1 Hardware Firmware controls Set Hold Temperature Opens the Set Hold Temperature dialog win dow User can then select from 20 to 70 C Selecting the OK button instructs the instrument to heat or cool and then hold the ingot at the desired te
22. eeeseeneeeeeeeeeneeeeeeeeaes 23 Chapter 4 HR 1 Instrument Control Software aisis to hanede a aaa a ee aaa a ai 25 Menu Bar Overview ccccccccceeeeeeeeeeencnneeeeeeeeeeeeeseecaceaeaeeeeeeeeeeeseseeeennnaeees 26 Setting Experimental 0 7 29 Starting the HR 1 Instrument control Software eeeeeeeeeeeeeeeeeeeeeen 29 29 0 Setting User 30 30 View Melting MOM syss arine AEAEE EENE ETENEE 31 Edit Melting Protocols 26 33 Multiple Sample Experiment cccccecceeeeeeeeeeecceeeeeeeeeeeeeeeeeseesnnaeees 33 HR 1 Instrument Introduction 4 Table of Contents LED MENU 2 edevanedeincsenes Tools venenatis FAS I gt Data Organization 0 eccceeeeeeeeeeeeeeeeeeeeeeeeeeneeeeeeeeeeees Melting a SAMPIe ee ceeeeeeeeeeeeeeeeeeeeeeeeeeeeeteeeeeeeeeeees Suggestions for Higher Throughput eceeeeeeeees Chapter 5 HR 1 Analysis Software IMtPOGUCTION oe cece Analysis Software Overview cccccecceeeeeeeteeeeeesenteeeereeaes Additional Sample and Graph Editing Options Data Analysis Introduction Opening the Analysis Software Importing Sample Data ccccceeeeeeeeeeeeeetceeeeeeeeeeees Normalizing Data eecceeceeeeeeceeeeeeeeneeeeeeeneeeeeeeeeeees X axis Temperature 80056 Additional Data
23. et Instrument 28 Resolution and Reproducibility AA A 9 RMA Number 00088 59 Seral POM gt lt 30 Set Hold Temperature 27 Set User Name 6 26 ererne nee ne 17 Software Installation 19 Table of Contents 3 Technical Support 2 Temp Shift ci2etacesitoudvncsentienexs 43 Troubleshooting 53 68 Index HR 1 Instrument Specifications 0 01 C to 1 C per second Ambient to 100 C 24 bit temperature and fluorescence analog to digital conversion 5 20 ul using Roche LightCycler capillary tubes Single sample capillary melter Typical 40 45 samples per hour with a 0 3 C ramp rate Dell Notebook Computer with Ethernet capabilities Windows based software includes control and analysis modules 4 5 5 12 inch 10 2 14 x 30 5 cm w h d 5 8 Ibs 2 65 kg 100 to 240 VAC 1 3 Amp 50 60 Hz universal power Linear Ramp Rate Temperature Range Data Acquisition Sample Volume Capacity Throughput Laptop Computer Software Size Weight Power Supply Idaho Technology Inc 390 Wakara Way Salt Lake City Utah 84108 USA ph 1 800 735 6544 801 736 6354 fx 801 588 0507 www idahotech com it idahotech com
24. f wild type sequence are distinguishable The following image shows a dual Domain Melting profile where heteroduplexes are pres ent in the high temperature and low temperature domains where both vari ants are readily discernible from fully base paired molecules Chapter 1_ HR 1 Scanning Principles 12 HR 1 Instrument Samples Multiple Domain Melting with 2 Different Mutations Multiple Domain Melting with 2 Different Mutations wigan 110 Mating Curves 17 Difference plot WT2 BAA Hett a AA 100 46138 AA Hett b 8 Hett b J 15 Het2 a f AA 90 Het2 a A 14 Het2 b 8 AA orl 13 80 12 11 10 70 60 Fluorescence 50 v 5 9 0 v 2 6 Wild Type 30 20 amp 8 amp 7 880 880 9 M 992 3 94 95 Temperature Temperature Domain Melting profiles from a fragment with a unique sequence variant in both domains Three samples WT Het1 and Het2 were analyzed in dupli cate Het1 contained a sequence variant in the low temperature domain while Het2 harbored a sequence variant in the higher temperature domain Because the melting profiles were grouped so tightly the variants were observed quite easily with a preliminary indication that each sample contained a unique variant in different portions of the fragment HR 1 Instrument 13 Instrument Specifications and Installation Chapter 2 CHAPTER Instrument Specifications and In
25. hat the capillaries fit firmly into the slot Care must be taken when inserting and especially when removing a sample as the capillaries become more fragile when they are heated Loading a Sample 1 To load a sample into HR 1 simply place the end of the cap illary into the hole on the top of the ingot and allow the capillary to seat itself i Manufacturer tolerances for the capillaries and heated ingots cause some capillaries to be loaded quite easily while others f Chapter 3_ _HR 1 Instrument Operating Instructions 4 HR 1 Instrument will need some amount of delicate pressure to be positioned correctly within the ingot Make sure the capillary is completely seated down in the ingot before the lid is shut 2 Shut the lid and use the software to begin melting Removing a Sample 1 When removing a capillary use two fingers to slide twist the capillary up and out of the ingot Take special care not to place excessive side to side directional force on the capillary as it is pulled out of the ingot as more of the capillary is removed from the ingot the less pressure is required to break the capillary with a sideways directional force HR 1 Instrument 25 The HR 1 Instrument Control Software Chapter 4 CHAPTER HR 1 Instrument 4 Control Software Introduction This chapter contains instructions on how to operate the HR i 1 instrument through the Control Software 6 HR 1 Ins
26. he HR 1 Instrument Control Software Chapter 4 Tools Menu The Tools menu accesses functions Rarer a ane ta AAEE pho E related to sample TUU fluorescent excita ns EA F tae Mamie so tion eon 2 After the HR 1 instrument Temperature is turned on the instru ment may be pre warmed by choosing Set Hold Temperature under the Tools menu A limit of 70 C has been set for safety If the instrument stops responding during a melt ing acquisition or at any other time the instrument i may be rebooted without Cancel power cycling or shutting Cancel down the software To complete this choose Reset Instrument under the Tools menu If the Reset Instrument function does not correct the problem turn the instrument off shut down the soft ware and restart software and or computer The name of the instrument can be edited or changed by selecting Instrument Name in the Tools menu Instrument Name Chapter 4 The HR 1 Instrument Control Software _ 36 HR 1 Instrument The Update Firmware option in Update Firmware Tools is available should Idaho Technology release firmware upgrades Further instructions for use of these functions will follow Fimware Version 20030717 through new product and upgrade announcements to HR 1 customers Firmware Image File Help Menu The HR 1 User s manual can be accessed in
27. ing Graph When the melting curve is finished the file is automatically saved and the HR 1 instrument runs the cool down fan until the ingot reaches the specified Cool Temperature When the cool down is complete the lid will unlock The lid may be opened and the sample removed at this point Follow the procedure to melt additional samples It will not be necessary to check all of the melt parameter fields if several samples are melting under the same conditions Type in the name of each new sample in the Start Melting window and select Start Melting for subsequent samples Suggestions for Higher Throughput The process of melting samples can be expedited by performing a test run through a temperature range of 70 C up to 96 C Following this protocol analyze the melting transition that occurs and choose a temperature range that will provide adequate data prior to and following the primary melting transition It may be helpful to use the derivative plot function to help pin point the maximal melting transition point indicated by the Tm of the result ing peak To determine the temperature range prior to and following the melt use the following calculation based on ramp rate 15 x Ramp Rate of C to set the acquisition temp prior to the initial melting transition of the fragment An example 15 x 0 3 ramp rate 5 Set acquisition tempera ture 5 prior to the melting transition and end melt 5 post transition Following this proce
28. ing the LightCycler amplification may be monitored in real time using fluorescence Channel 1 Amplification may be performed in other reaction vessels however the specimen must be transferred to a Roche LightCycler capillary prior to analysis in HR 1 Amplification reac tions of 10 ul are effectively analyzed by HR 1 and this volume is recom mended however reaction volumes of 5 20 ul can also be used A small volume oil overlay may provide even greater precision when added prior to amplification Preparation of a DNA Sample Numerous methods exist to prepare DNA from a variety of sources Any variation in salt concentration between samples will adversely affect preci sion of the melting curve To a lesser extent variation in the DNA concen tration can also affect melting profiles When assaying groups of samples it is advisable that they be adjusted to a uniform concentration in the same buffer Variable results have been observed with samples that have been archived for significant periods of time It is strongly recommended that archived DNA samples be re purified eluded in a standard buffer and quan tified before use with the HR 1 system Following re purification archival specimens perform as well as newly prepared specimens Amplification Amplification can be performed any number of ways in the presence of LC Green dye It must be robust and performed prior to analysis on the HR 1 Amplification may be done in Roche capil
29. ion heteroduplexes are identi fied by a change in shape of the melting curve from fully base paired wild type samples Distinguishing factors include the slope of the primary melt ing transition the lowest temperature at which heteroduplex samples devi ate from wild type samples and the temperature at a given fluorescence level Although quantitative measures can be devised observation of the entire curve and comparison to wild type is the best method for identifying hetero duplexes Chapter 5 _ HR 1 Analysis Software 6 HR 1 Instrument Opening the Analysis Software To open the HR 1 analysis software select the HR 1 melt Wa analysis tool desktop icon or choose Melting Analysis under the Melting menu in the HR 1 Instrument Control HR 1 Melt software A full screen window will open along with a Analysis Tool browsing window Importing Sample Data A window will appear prompting Open the Folder your Files are in and Press Select Cur Dir bottom right selection of analysis files Browse to the location of the melting files and choose the Select Current Directory This will open a sample list window in the HR 1 analysis pack age All vs2 files in the directory are selected for analysis by default If only a subset of the samples is to be analyzed select the samples to ana lyze while holding the Shift key for blocks of samples or choose individual samples to view Select Continue when finished
30. ique characters in the sam ple names list all common characters in the sample names will be displayed in the graph title Change Changes sample names Export Graph Data Creates a tab space delimited text file for use in external database or spreadsheet applications Print Print Window Opens the systems default printer window Page Setup Change print parameters and layout Exit Close analysis software Edit Edit functions are operable only if graph tools have been used in the analysis Undo Move Undoes movement caused by use of the graph tools Redo Move Redoes graph movement after use of Undo Move Select Original Data Default setting uses all time temp fluorescence data logged by HR 1 Limit by Time Temp Allows user to define the time temperature rang es to analyze Melting Curves Default setting shows data as a fluorescence vs temperature plot Kinetics Shows data as a fluorescence vs time plot Time Temp Plots the temperature vs time gives an indication of tem perature control HR 1 Instrument 43 HR 1 Analysis Software Chapter 5 Analyze Normalize Normalizes fluorescence data before and after the melting transition Temp Shift Force all samples to overlay over a defined temperature fluorescence range Difference Plot Graphical plot of the difference between samples at each data point Derivative Plot Graphical plot of the rate of fluorescence chang
31. is provided as a standardization tool when a MA HR 1 Instrument 49 HR 1 Analysis Software Chapter 5 true standard sample has been run for comparison purposes As with nor malization there are no set rules for cursor placement but experimentation will quickly provide the necessary experience to become proficient in X axis temperature adjustment Additional Data Display Options While Normalizing and X axis tem Hi perature shifting are the only steps required for most analysis there are two additional options for viewing data in different formats which are useful in making the determination of whether or not a sample contains a sequence variant or whether the sequence variant observed in one sample is the same as that observed in another sample Fluorescence F1 Melting curves of 2 heterozygous samples with different mutations in the same target sequence Some differences are observed in the melting curves but they are still quite similar Difference Plot The Difference Plot function provides p a graphical method of looking at the temperature difference between two oss DualHett Data Adjust Vertical Cursors or several samples throughout the 9 F selected temperature range Under the Analyze menu select Difference Plot A window will open with the 6 samples listed in the upper left por tion Select a reference sample It is represe
32. laries or transferred to them post amplification for analysis on the HR 1 It is possible to use an oil over lay with the amplification and leave it in place for the analysis on the HR 1 Oil overlay has been shown to be beneficial with repetitive melts on the HR 1 Instrument 23 HR 1 Instrument Operating Instructions Chapter 3 same sample Melting profile similarity between like samples is critical to the ability to dis tinguish differing melting profiles of samples harboring a sequence variant If the melting profiles of different samples show more variation than is desir able it may be useful to add a mineral oil overlay 2 5 ul The mineral oil should be applied prior to amplification Because of the exceptional resolu tion of HR 1 small irregularities between samples related to evaporation condensation during amplification or during a melt on HR 1 may have an effect on the salt or dsDNA concentration post amplification between sam ples This may cause artificial differences between melting curves of per fectly homologous samples A small volume mineral oil overlay will prevent this from occurring and has the effect of tightening up the melting profiles to a degree that it is sometimes even possible to distinguish a homozygous base change between samples Loading and Unloading a Sample All melting curves must be performed in Roche LightCycler capillaries The heated ingot in the HR 1 instrument is designed so t
33. mperature Only available when the instrument is not melting Chapter 4 The HR 1 Instrument Control Software _ 8 HR 1 Instrument Instrument cannot hold a temperature lower than ambient setting a hold temperature lower than ambient will cause the cooling fan to run resulting in an error prompt reading Unable to reach xx degrees cooling aborted xx is the selected cool temperature The instrument will not accept hold tempera ture greater than 70 C for safety reasons Reset Instrument Re initializes the firmware and internal hardware components Accessible when instrument is not melting Instrument Name Records a unique name for the instrument Instrument name is logged in data files Update Firmware Opens the Update Firmware Dialog Accessible when instrument is not melting Used only when new firmware is pro vided by Idaho Technology Done Closes the Update Firmware Dialog Browse Opens an Open File dialog to find the firmware HEX file Refresh Refreshes the displayed instrument version Update Updates the instrument with the selected firmware HEX file Remove File Removes the currently selected file from the drop down list Help Accesses the User Help Functions About HR 1 Opens the About Dialog OK Closes the About Dialog Reset Re initializes the firmware and internal hardware components Accessible when instrument is not melting User Manual Accesses the electronic version of
34. n the lid with any force if the latch is stuck This can bend the latch and cause it to become inoper able Instrument does not Respond to Computer Prompts Solution 1 Check the Com Port selection to verify the right com port has been chosen Incorrect com port selection results in an inability of the com puter to communicate with the HR 1 Solution 2 If the instrument stops responding during melting acquisition or at any other time it is possible to reboot the instrument without turning the instrument off and on or shutting down the software To reboot the HR 1 select Reset Instrument under the Tools menu If the Reset Instrument function does not correct the problem turn the instrument Chapter 6_ Troubleshooting 6 HR 1 Instrument off shut down the software and re initialize the software and or reboot the computer Power Light does not Light up when Power is Turned on Solution Unplug the HR 1 from the wall socket Disconnect the Serial cable from the back of the panel Check fuses on the back panel of the instrument If a fuse is blown replace with 1amp 250 volt 3AG fuses this information is also located on the back serial plate using the fol lowing diagram and description The fuse assembly is located next to the power switch Place a flathead screwdriver in the small slot to pop the fuse assembly out of the mounting hole The fuse assembly will pop up when the plas tic latch is released
35. ngle peak excitation 440 470nm and Introduction 6 HR 1 Instrument emission 470 520nm spectra The fluorescent dye LCGreen is includ ed in the amplification reaction and analysis is performed in a closed tube system HR 1 in combination with LCGreen generates melting curves that exploit the subtle differences between heteroduplex and fully based paired wild type species The instrument can run 40 45 samples per hour post amplification with a 0 3 C sec ramp rate Up to 32 samples may be viewed concurrently with the analysis software The HR 1 system is the only commercially available closed tube homogenous reaction platform to scan for heteroduplexes HR 1 Instrument 7 HR 1 Scanning Principles Chapter 1 CHAPTER Principles of Mutation Scanning and Genotyping Introduction This chapter explains general concepts of mutation scanning and genotyping This includes consideration of heteroduplex DNA molecules characteristics that facilitate them being distinguished from fully base paired DNA molecules and how LCGreen and HR 1 are used to detect hetero duplexes Genotyping of different homoduplexes by melting temperature is also considered Heteroduplex Detection A gene which is heterozygous at a particular locus will upon amplification create two unique double stranded DNA molecules If the two molecules differ at a single site within the sequence the products Heterozygous Sample can schemati
36. nstrument 31 The HR 1 Instrument Control Software Chapter 4 Melting Menu To start a melting curve select Start Melting from the Melting menu The Start Melting window will appear with the currently select ed melting protocol and the default file name for the sample to be melt ed The default file name is a Date Time stamp but can be changed by typing in the desired file name If the currently selected file name is appro priate load a sample shut the lid and choose Start to begin the melting curve To edit or verify that the currently selected melting protocol is correct select the Edit Protocol button To select a different melting protocol use the pull down menu to choose the correct protocol A new Melting Protocol can be created by choosing the New Protocol button Melting Protocol Name Note It is recommended to melt the first 0000000000 0 sample of a new experiment in order to Bamp Rate 030 verify the pathway to which the file will be _ I Repeat Melting sea The Edit Protocol button opens n a a dialog window with the parameters for Cool Temperature fs Hold Cool Temp the currently selected melting protocol Acquisition Stat Temperature B4 displayed in the melting parameter fields Directo CAProgramFies idaho Tec Browse Melting parameters can be modified in this window or the pull down menu can be used to select a preset melting proto 3 Fluorescence 1 70 col saved
37. nted by the horizontal line in the graph area The differ 9 ence between each sample and the 7 0 0 09 reference sample is calculated and Chapter 5 _ HR 1 Analysis Software 0 HR 1 Instrument displayed for each data point in the comparison The Y axis is auto scaled based on the largest difference between samples The Difference Plot may be a useful tool to determine whether or not a suspected sequence variant in one sample is potentially a different sequence variant than that observed in another sample It can be difficult to make this determination by look ing only at the melting curves The Difference Plot and Derivative Plot described below provide displays that may allow distinction between 2 dif ferent sequence variants Essentially subtle differences between variant sample melting curves are magnified and easily observed using these two graphical display options Samples Het 18 Het 28 Het 38 18 28 38 Wild Type Samples Het 18 Het 28 Het 3 f WT 18 Heteroduplex WT 28 WT 38 Fluorescence Fluorescence 81 82 79 81 82 Temperature Temperature Melting curves left panel and Difference Plots right panel of 3 different Heterozygous samples with the same mutation Note how the heteroduplex curves are identical in both graphs as are the curves of the WT samples 80 HR 1 Instrument 51 HR 1 Analysis Software Chapter 5 Derivative Plot
38. on and eliminates the potential for dye redistribution during the melt The ability to use saturating levels of LCGreen and the tightly controlled temperature capabilities of HR 1 allow differentiation of the subtle fluorescence changes during the melting transition that occur in a sample containing heteroduplex species Genotyping with HR 1 and LCGreen HR 1 and LCGreen were designed to scan for heteroduplexes within amplification products The high resolution of the system allows differ ent heterozygotes to be distinguished from each other That is different heterozygotes can be genotyped if the controls are available For example hemoglobin Hgb AS AC AE and AS were all distinguishable from each other and the wild type AA in a 110 bp fragment of R globin Clinical Chemistry 2003 49 853 860 Furthermore it is often possible to distin guish different homozygote base pair changes for example Hgb AA SS and CC However some homozygous differences are difficult to detect A to T and G to C SNPs When the differences between homozygous geno types is small the detection rate will increase when smaller products are analyzed Product Size and Domain Melting The size of an amplification product that can be successfully analyzed on HR 1 is a variable whose upper limit has not been resolved and may not be governed by a defined set of rules When products up to 600 bases were systematically studied all heteroduplexes were detec
39. r 2_ Instrument Specifications and Installation 0 HR 1 Instrument HR 1 Instrument 21 HR 1 Instrument Operating Instructions Chapter 3 CHAPTER HR 1 General Operating Instructions Introduction This chapter details the steps for preparing samples and loading them into the HR 1 for analysis General Considerations The HR 1 LCGreen platform directly analyzes the melting behavior of double stranded DNA LCGreen dye binds to any dsDNA present Therefore care in preparation of the DNA sample to be assayed is critical for generating optimal results Factors that influence results obtained using the HR 1 include the nature of the DNA specimens being analyzed and fac tors influencing thermal denaturation of a DNA fragment Analysis on the HR 1 requires that defined DNA fragments are present usually produced by amplification The buffer and ionic strength are especially important and must be controlled from sample to sample The HR 1 analyzes specimens in Roche LightCycler capillary tubes These tubes are designed for the LightCycler instrument and are also com Chapter 3 _ HR 1 Instrument Operating Instructions 22 HR 1 Instrument patible with the Idaho Technology RapidCycler 2 and R A P 1 D systems A clear benefit of amplification in Roche LightCycler capillaries is that after amplification the tube may be transferred directly to the HR 1 for analysis Additionally when us
40. rature range and up to 32 samples may be viewed concurrently with the analysis software There are three indicator lights on the front of the instrument All three lights come on when the instrument is turned on During instrument opera tion the lights function as follows Steady Green light indicates instrument power is on Flashing Red light indicates instrument is melt ing a sample and the lid is locked shut Flashing Amber light indicates instrument is unlocked and ready to analyze another sample 11 Instrument Specifications and Installation 16 HR 1 Instrument Components of the HR 1 System HR 1 Instrument Package Laptop computer pre loaded with HR 1 Instrument Control and Data Analysis software Software installation CD HR 1 Startup Kit includes Manual and Cables HR 1 Tool Kit Power Kit 1 Box Roche LightCycler capillaries 96 capillaries HR 1 LC Green Chemistry Kit only available with HR 1 instrument purchase 100 ul of LCGreen 10x dye 1 ml 10X Buffer with BSA 10 mM 2 1 ml 10X Buffer with BSA 20 mM MgCl2 1 ml 10X Buffer with BSA 30 mM MgCl2 1 ml of Enzyme diluent 1 ml of 10X BSA 2 5 mg ml 400 1 000 and 10 000 LCGreen Reaction Kits are available from Idaho Technology Reagent Recommendations LCGreen dye is to be used at 1X final concentration BSA is to be used at 1 10 concentration 0 25 mg ml This is to help avoid adsorption of nucleic acids to glass capillaries
41. rn 8 Decontamination Forms 6 HR 1 Instrument HR 1 Instrument 67 Index A H Amplification 10 66 1618700 22 8 Analysis Software 46 Heteroduplex detection 7 Auto Set Fluorescence 57 Heterozygous 22 ciceaaiceeicn 7 Higher Throughput 39 0 Cleaning the 90 53 I Cl ar Dalase 26 Importing Sample Data 46 55 Installation 000 13 Control Software 25 Introduction 5 Customer Support 2 K D KINetics 42 Data Display Options 49 Data Organization 36 L Decontamination 58 59 LCGreen dsDNA binding Derivative Plot 43 51 a ee ek ee 9 Difference Plot 43 49 Loading a Sample 23 Domain Melting 10 M F Melting 80 26 FUSES 8 56 Melting Protocols 27 33 Multiple Sample Experiments O eeaeee O eB eeeetaapsueccetan 33 Genotyping seese 10 Mutation Scanning and Graph Editing Options 45 Genotyping scdeasconsvoinnscecentass 7 HR 1 Instrument User Name Profile 29 Wild type eeeer eererrerer rere errer rere 9 X axis Temperature Adjustment 48 Normalizing Data 47 Operating Instructions 21 Packaging and Shipping 60 Pause Data sissies 26 Preparation of a DNA Sample 0 22 Product 10 Removing a Sample 24 Res
42. ry Information This symbol warns the user to operate the machine according to the instructions provided in this manual If used otherwise a potentially hazardous situation could result This symbol is used to label potentially hot instruments surfaces This symbol highlights user tips to operate The HR 1 instrument more efficiently Note Always maintain The HR 1 instrument in good working order If the instru ment experiences an extreme environmental event return it to Idaho Technology for a complete service inspection See Appendix A for return instructions If The HR 1 instrument is used in a manner other than described in this manual it may impair equipment protection and performance Introduction 2 HR 1 Instrument Customer and Technical Support Reach Us On the Web Idaho Technology s Web site is http www idahotech com Order LCGreen Reagent Kits at http www itbiochem com We encourage users to visit our Web site for answers to frequently asked questions The HR 1 instrument manuals parts and accessories are avail able online Reach Us By E mail Contact Idaho Technology by e mail in the following areas support idahotech com Technical Support it idahotech com General Support Reach Us By Phone Technical support is available during the following times 8 a m to 5 30 p m Mountain Standard Time For technical support call 1 800 735 6544 United States and Canada 1 801 736 6354 Utah IDD Prefix 1
43. sing Temp Shift under the 8 AAA VW Analyze menu A window will open displaying the normalized curves in Babe cine i the top panel and a preview of the temperature adjusted curves in the 4 lower panel Temperature adjust ment forces each curve through the same temperature range as defined by the placement of the horizontal cursors This is useful for heteroduplex analysis in that differential melting curve shapes are more easily distinguished allowing for positive identifica tion of a sample harboring a sequence variant The default placement of the horizontal cursors shown in the middle graph is adequate in most cases If the cursors need adjustment left click the mouse and hold on the cursors to move them It is recommended that the cursors be kept as close to the bottom and as close to each other as possible thus ensuring the curves will be adjusted and forced through the highest temperature point of the melting transition Use the bottom graph to judge the effect of cursor adjust ments A list of sample names is shown with a graphical representation of the data points included in the current range defined by cursor placement for each sample in the middle panel Select from this list the sample to rep resent the reference curve All other curves will be fit to this reference sample curve Choosing a different reference sample for this analysis has little effect on the outcome but
44. stallation Introduction This chapter contains general and technical information about the HR 1 instrument including specifications installation requirements and installa tion instructions It is included for those who will install and perform routine maintenance on the HR 1 instrument Using the sys tem improp A erly may compromise performance or permanently damage the instrument Idaho Technology Inc Lid Catch Solenoid 222009920800 209 11111 7 1 9 Cooling Fan Ingot 5 2 O HR 1 Instrument 14 Instrument Specifications and Installation Chapter 2 HR 1 Instrument 15 Instrument Specifications and Installation Chapter 2 The HR 1 Instrument The HR 1 instrument consists of a single heat controlled ingot machined aluminum cylinder positioned above an optics block that functions as an excitation and data collection source Sample analysis is performed in a Roche LightCycler capillary tube While there is a clear advantage to per forming amplification in a capillary tube amplification may be performed in other containers and subsequently transferred to a Roche capillary for anal ysis The fluorescent dye LCGreen is included in the amplification reac tion The HR 1 platform is the only homogenous system to scan for hetero duplexes A single sample may be scanned in 30 120 seconds dependent on ramp rate and tempe
45. t them All of the curves should appear in the lower panel preview area Positioning of the cursors during the normalization process is critical to further analysis of the samples If samples did not amplify or no template controls were present these samples will not normalize properly Eliminate these samples from consideration by selecting Delete Some Bad Samples or Delete All Bad Samples It is possible to exclude bad samples by simply not select ing them for the final analysis There are no set rules for positioning the cursors Brief experimentation of different cursor placements will display the functioning of the cursors and the different effects As a general rule it is advisable to include a larger temperature range between each set of cursors as ramp rate increases At a ramp rate of 0 3 C per second a temperature range of at least 0 5 C between cursors is desirable After the data has Chapter 5_ HR 1 Analysis Software 8 HR 1 Instrument been normalized all samples will have a flat slope before and after the melt This makes further analysis of the melting portion of the curves easier to interpret X axis Temperature Adjustment The most common application on the 8 HR 1 is scanning amplified products Samples gt mN A for heteroduplexes For this applica tion X axis temperature adjust of data a is necessary following normaliza tion Temperature adjust the curves by choo
46. table when oil was used Amplification products in the range of 100 250 bases can be rou HR 1 Instrument 11 HR 1 Scanning Principles Chapter 1 tinely analyzed on HR 1 and exhibit a single clean melting transition of the entire product Localized regions excessively rich or poor in G C base pairs resulting in correspondingly poor or rich A T regions respectively may experience Domain Melting Domain Melting describes a localized region within a fragment that denatures melts at a temperature below that of the remaining portion of the fragment While both targets were similar in overall G C content the fragment showing Domain Melting characteristics has an internal region that is relatively rich in A T base pairing Melting Curves Single Domain melt Melting Curves Dual Domain melt 900065 BAW Fluorescence Fluorescence 84 Temperature Temperature The figures above show melting profiles from different target sequences with a single melting domain and a fragment containing high temperature and low temperature melting domains Domain Melting is most likely to be observed in amplification products over 200 bases but can also be seen in fragments as small as 100 bases The presence of multiple individual melting domains does not preclude effective analysis with HR 1 The primary concern is that melting profiles of different samples align such that differences between samples harboring a sequence variant and those o
47. tain an RMA Number The first step in returning an instrument to Idaho Technology is to obtain an RMA number from our Service Department Fill out the supplied fax forms 2 pages and fax them to Idaho Technology at 801 588 0507 Our Service Representative will contact you with an RMA number and further instruc tions If your machine is still under warranty please supply the purchase date and serial number if your machine is out of warranty please supply a blank PO for the repair charges Decontamination all Returned Equipment Equipment returned to Idaho Technology for repair and other reasons must be handled in a manner which will ensure minimal risk to the personnel involved in packing the unit for return and those responsible for receipt unpacking and processing at Idaho Technology Inc To ensure there is no loss of data any computers being retuned must be backed up prior to shipment Idaho Technology is not responsible for any lost data Decontamination The person responsible for the return must thoroughly decontaminate the unit wiping it down with 0 5 NaOCl chlorine bleach diluted 1 part bleach to 9 parts water Use care not to mix with other chemicals mixture with acid will liberate chlorine gas Since the solution is somewhat caustic gloves and a face shield are recommended when preparing and using the solution When corrosion by NaOCl may be a problem a flush with water is permissible after the NaOCl has remained in
48. the User s Manual Idaho Technology Technical Support Links Users to the Idaho Technology Technical Support by email compatible with Mozilla and Outlook not compatible with Outlook Express HR 1 Instrument 29 The HR 1 Instrument Control Software Chapter 4 Setting Experimental Conditions Starting the HR 1 Instrument Control Software Confirm the HR 1 instrument control software is loaded onto the hard drive the instrument is connected by the serial cable and the power on the HR 1 instrument is on If any of these items are not in place refer to Chapter 2 1 a 1 To start the HR 1 Instrument Control software by dou Bi ble clicking on the HR 1 Instrument Control icon on Moten the desktop Control 2 The Enter Username dialog x window will appear Use the pull down menu to select the User Name This will allow fo Cancel protocols stored under the User Name Setting User Name Profile To change the User Name without shutting down the instrument or software select Set User Name from the File menu and change the user on the pull down menu To Create a new user simply type the new User Name in the Set User Name window To delete a User Name select the User Name to be deleted from the pull down menu and choose Remove User Deleting a User Name results in all protocols saved under that User Name also being deleted Protocols saved as Global will remain even if all users are removed
49. ther experimental conditions Melting acquisition can be stopped at any time by selecting Abort Melting from the Melting menu Data is continuously logged during a melting proto col and any data that has been logged will be saved under the file name Chapter 4 _ _ The HR 1 Instrument Control Software 34 HR 1 Instrument Melting protocols can be created and edited by choosing New Protocols under the Melting menu To edit an existing protocol right click on the pro tocol and select Edit Melt Protocol To create a new protocol right click on the user name for the person designated for the new protocol Under the Melting menu the analysis software package can be opened by selecting Melting Analysis LED Menu The HR 1 instrument can be used as a real time fluorimeter to check the fluorescent status of a sample under different simula aut LED power settings This func 30 tion gives an accurate measure of the strength of fluorescent i cation of the fluorescent acquisition parameters to set in the melting param eter fields see Melting menu above To turn the LED on select Turn LED Power LED On under the LED menu Click on Adjust LED Power under the LED menu to view the effect of different LED power settings on the fluorescent signal in a sample The LED is automatically turned on when this option is used To turn off the LED choose Turn LED Off under the LED menu HR 1 Instrument 35 T
50. tive rights to the computer are required to install and run the software package on other computers The software installation will require up to 19 MB of disk space All HR 1 soft ware must be run at a screen resolution of 1024 x 768 HR 1 software package All software for running the HR 1 and analyzing data is pre installed on the HR 1 laptop The software is located under the Idaho Technology folder in the Programs directory An installation CD is provided for re installation or installation of the analysis software package on multiple computers Be sure to back up data before re installation of software To Install the Software Insert the HR 1 CD into the CD drive The installation wizard will automati cally start up If the installation wizard does not prompt browse to the CD drive select the HR 1 software file and choose the setup exe file After selecting the installation location the Installer will present three options Typical Compact and Custom Typical Installation This will install all the software needed to run the HR 1 Instrument and analyze the data it produces Compact Installation This option will only install the HR 1 Instrument Control software This option is for users where HR 1 data analysis is to take place on a different computer than the one controlling the HR 1 Instrument Custom Installation Allows the user to choose individual components of the HR 1 software to install on the system Chapte
51. tocol to examine that all melt parameter fields are cor rectly filled To melt more than one sample under the same conditions check the Repeat Melting box By checking the Repeat Melting box the Start Melting window will automatically open after the a melting protocol is completed and the ingot has been cooled to the specified temperature Check the Hold Cool Temp box for the instrument to maintain the cool down temperature between melting acquisitions There are two options for fluorescence acquisition in the Edit Protocol win dow Select the Auto Set box to automatically adjust the LED power to achieve a specified level of fluorescence from the sample Uncheck the Auto Set box to use a standard LED power level on all samples Confirm the path where the sample file will be saved choose the Browse button to change the path and Save when the desired path is found The Start Melting window will return upon completion of Protocol editing choose OK or cancel HR 1 Instrument 39 The HR 1 Instrument Control Software Chapter 4 Name the sample in the file name text area load the sample capillary in the instrument close the lid and select Start Melting The lid will lock at this time and cannot be opened until the ingot is cooled to the specified Cool Temperature maximum cool down temperature is 70 C During melting acquisition it is possible to change the display window to dis play the Real Time Graph or the Melt
52. trument 1 To open the software locate the software icon on the 2 Desktop 2 Using the mouse double click on the icon which will take b you to the main ot screen Chapter 4 The HR 1 Instrument Control Software 6 HR 1 Instrument Menu Bar Overview File Options access basic user interface functions Serial Port Designates computer serial interface to the HR 1 Com1 Com2 etc Set User Name Accesses Enter Users menu for different operators Pull Down Selection menu Allows user to select user name and add new user names OK Accepts pull down menu selection Cancel Cancels out of Set User Name window Remove User Removes selected user from pull down menu Exit Exits from HR 1 instrument control software View Alternates between view panes and data streaming options Real Time Graph Checkable option to monitor HR 1 in real time Melting Graph Checkable option to monitor HR 1 recorded experi mental fluorescent trace Pause Data Pauses and re starts real time graphing updates Fluorescence and temperature data is continuously displayed in the upper left status bar Clear Data Clears stored temperature and fluorescence data on both graphs Accessible when instrument is not melting Melting Accesses melting experiment conditions and controls Start Melting Opens Start Melting window Start Begins selected melting protocol The instruments lid must be clos
53. under the current user s name Excitation Power 0 100 50 VV Auto Set i The HR 1 Instrument Control Software 32 HR 1 Instrument Melting Protocol Name Defines protocol name which must be unique to the User and Global list Repeat Melting Automatically prompts the Start Melting window with a previous protocol for repetitive runs Ramp Rate Rate of temperature change for the melting profile 0 01 1 0 C per second Final Temperature Ending High temperature melting curve 100 C max Cool Temperature Temperature the HR 1 will cool to after the Final Temperature is reached Hold Cool Temp Selectable feature to hold HR 1 at a staging temper ature for repeated melts to aid in throughput 20 70 C programmable range Acquisition Start Temperature Temperature when the HR 1 melting profile begins acquiring fluorescent data Directory Location where melting data files are stored Data files have a vs2 extension Excitation Power LED power setting for the melt protocol Auto Set The HR 1 automatically determines the optimal starting LED power setting based on the target fluorescence Target Fluorescence Target scale setting for Auto Set fluores cence 99 max HR 1 Instrument 33 The HR 1 Instrument Control Software Chapter 4 Edit Melting Protocols The Melting Protocol Name Temperature transition rate Ramp Rate beginning Cool Temperature and ending temperat
54. ures Final Temperature the location to which the melting file is saved vs2 exten sion and the Target Fluorescence can be modified in this menu Multiple Sample Experiments If several samples are to be melted during the same session check the Repeat Melting and Hold Cool Temp boxes This will automatically bring up the Start Melting window with the file name highlighted following each melting protocol and will hold the ingot at the specified cool down tempera ture until the next melting protocol is started Anew sample name can be entered while the previous sample and ingot are being cooled When editing or creating a new melting protocol it is recommended that the Cool Temperature be set at least 10 degrees below the Acquisition Start Temperature to allow the instrument time to gain sufficient control of the heated ingot prior to the initial acquisition of data If the Auto Set check box is checked the instrument will automatically adjust LED power to acquire the desired fluorescence target level from each sample If the Auto Set box is unchecked the user specifies a standard LED power that will be used on each sample Checking the Auto Set box is advisable when the assay conditions have been optimized and the user wishes to perform the final analysis on each sample Leaving the Auto Set box unchecked may be useful when determining how much fluorescence is present in individual samples relative to each other or compared to o
55. use the delivery of products which do not appear to have been properly decontaminated If the equipment was used with or around radioactive material the signature of the safety officer is also required Signature Date Appendix A_ Return 8 Decontamination Forms 64 HR 1 Instrument Decontamination Labels Pg 1 of 2 Fill out the decontamination Labels and affix one to the product and the other to the exterior of the shipping carton Failure to decontaminate before shipping to Idaho Technology Inc will result in the immediate return of the unit at your expense This Product has been decontaminated per Return Equipment policy Decontamination Notice S 4 2 a N 5 5 lt x 5 5 92 4 gt D Q O 5 5 8 e Oo B Customer Name Customer Address HR 1 Instrument 65 Return amp Decontamination Forms Appendix A Decontamination Labels Pg 2 of 2 Fill out the decontamination Labels and affix one to the product and the other to the exterior of the shipping carton Failure to decontaminate before shipping to Idaho Technology Inc will result in the immediate return of the unit at your expense This Product has been decontaminated per Return Equipment policy Decontamination Notice S 4 2 a N 5 5 lt x 5 5 92 4 gt D Q O 5 5 8 e Oo B Customer Name Customer Address Appendix A_ Retu
56. ver on and carefully remove the cover to expose the ingot and the area around the ingot with a swab and 10 bleach solution ingot to the interior of the HR 1 instru ment with the hex head driver tool supplied in the HR 1 Tool Kit and carefully lift the ingot off the two positioning posts that protrude through the ingot s circuit board of the ingot with the diluted bleach solu tion Clean the interior of the ingot with the small wire brush and diluted bleach solution Repeat as necessary lens under the ingot with a diluted bleach solution followed by water Use the small Phillips head screwdriver Carefully and thoroughly clean the ingot Detach the connector running from the Remove the two small hex head screws Clean the conical portion of the bottom Using a lens cleaning cloth clean the 1 HR 1 Instrument 55 Troubleshooting Chapter 6 8 Place the ingot over the positioning posts and re seat the assembly 9 Re attach the connector to the ingot 10 Replace the two small hex head screws but do not tighten either of them down until both screws have been started in k their respective holes Care should be taken NOT to over tighten these screws 11 Replace the plastic cover and the two small Phillips head screws Lid will not Unlock after Run Solution Tap gently on the top of the instrument Listen for the latch to release and open the lid Do not attempt to ope
57. yles for easier discrimination between samples Black and White Displays samples as a spectrum of thin to thick gray to black lines User Select a previously saved User Style see Save User Style below Replicates Duplicates Displays samples as duplicates of each other Sample 1 and 2 Sample 3 and 4 etc Triplicates Displays samples as triplicates of each other Quadruplicates Displays samples as quadruplicates of each other By Last Alphanumeric Uses the last alphanumeric character to identify sample replicates HR 1 Instrument 45 HR 1 Analysis Software Chapter 5 Save User Style Saves sample editing changes for the current analy sis About HR 1 v1 1 Shows the current version of analysis software Additional Sample and Graph Editing Options Right click on any sample color line to change color line style line width point style etc Select the first or last number on either the X or Y axis to change the scale of the axis Select a Graph Tools icon from the Graph Tools palette located just off the lower left corner of the graph and and drag within the graph This also activates the Edit functions Data Analysis Introduction The approach to analysis of data from the HR 1 will be based on the spe cific application Whole amplicon melting curve analysis most commonly is used to scan for heteroduplex formation It can be used for genotyping as well When scanning for heteroduplex format

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