Patent Publication Number: US-2012043479-A1

Title: Normalization of Biomolecules

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application claims priority to U.S. Provisional Patent Application No. 61/374,535, filed Aug. 17, 2010, the entire disclosure of which is incorporated by reference. 
    
    
     FIELD OF THE DISCLOSURE 
     The present disclosure relates in part to normalization of biomolecular components such as DNA, RNA, protein, starch and lipids concentrations. More particularly, the present disclosure relates in part to the processing and normalization of multiple samples, including DNA samples. 
     BACKGROUND OF THE DISCLOSURE 
     Accurate quantification and normalization of biomolecular components is important for further analysis of the samples. For example, quantification and normalization of genomic DNA from tissue samples is important for, for example, conducting DNA based analyses used in sequencing, southern analysis, marker assisted breeding, zygosity testing, and adventitious presence testing. Manual normalization of DNA samples may be time consuming, and may be a bottle neck in a laboratory testing DNA samples. In a 96-well plate, 96 samples may need to be analyzed and normalized, so that the samples are each within a range of concentration for proper analysis. Accidentally normalizing the wrong well or contaminating a well is a possibility when normalizing DNA manually, leading to errors and additional work to re-run the samples. 
     SUMMARY 
     The present disclosure provides systems and methods for normalization of multiple samples, including DNA samples. 
     The subject invention allows for decreased human error, decreased ergonomic concerns, and an increase in throughput. The subject invention provides systems and methods that allow for automated normalization of samples in multi-well plates. 
     According to the present disclosure, a method for normalizing samples is provided. Some preferred methods comprise determining florescence data of one or more wells on a plate; electronically, using a processor, calculating dilution data for one or more of the wells on the plate based at least in part on the florescence data; and adding a liquid to the one or more wells on the plate based at least in part on the dilution data. 
     Additional features and advantages of the present disclosure will become apparent to those skilled in the art upon consideration of the following detailed description of the illustrative embodiments exemplifying the best mode of carrying out the invention. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The detailed description of the drawings particularly refers to the accompanying figures in which: 
         FIG. 1  is a component view of an exemplary normalizing system according to an embodiment of the present disclosure; 
         FIG. 2  is a component view of the normalization system  107  of  FIG. 1  according to an embodiment of the present disclosure; 
         FIG. 3  is a flowchart showing a method of normalizing samples according to an embodiment of the present disclosure; 
         FIG. 4  is an exemplary representation of a 96-well plate, showing three conditions according to an embodiment of the present disclosure; 
         FIG. 5  is an exemplary input dataset according to an embodiment of the present disclosure; 
         FIG. 6  is an exemplary output dataset according to an embodiment of the present disclosure; and 
         FIG. 7  is an exemplary view of settings for customization of the normalization calculations according to an embodiment of the present disclosure. 
     
    
    
     Corresponding reference characters indicate corresponding parts throughout the several views. The exemplifications set out herein illustrate exemplary embodiments of the disclosure and such exemplifications are not to be construed as limiting the scope of the disclosure in any manner. 
     DETAILED DESCRIPTION OF THE DRAWINGS 
     The embodiments of the disclosure described herein are not intended to be exhaustive or to limit the disclosure to the precise forms disclosed. Rather, the embodiments selected for description have been chosen to enable one skilled in the art to practice the subject matter of the disclosure. Although the disclosure describes specific configurations of a normalization system  107 , it should be understood that the concepts presented herein may be used in other various configurations consistent with this disclosure. 
       FIG. 1  shows a component view of an exemplary normalizing system according to an embodiment of the present disclosure. 
     A plate may be prepared shown in box  103 . The plate may contain a number of wells, and one or more samples may be disposed in each of the wells. The samples may be any type of biomolecular component such as, for example and without limitation, one or more DNA samples, and may be prepared according to any known methods or practices. In one embodiment, the plate is placed into or provided to a spectrophotometer  105 , although any type of machine or process to provide an analysis of a sample may be used. In one embodiment, the spectrophotometer  105  may be a Spectra Max GEMINIS XS microplate fluorometer from Molecular Devices (Sunnyvale, Calif.). The spectrophotometer  105  shines light of any spectrum or spectrums onto the plate, or onto one or more wells of the plate. The light is transmitted through the sample and to a photodetector or other measurement instrument, where the samples florescence is measured. The spectrophotometer  105  may perform additional calculations to determine, for example and without limitation, the concentration of one or more components of the sample. The spectrophotometer  105  may produce data. The data may be in the form of, for example and without limitation, well location information, florescence information corresponding to the well location, or other calculations related to the florescence information. The plate may be removed from the spectrophotometer  105 . 
     The data from the spectrophotometer may be provided to the normalization system  107 . The data may be provided by a network or a dedicated connection between the spectrophotometer and the normalization system  107 , or by a removable storage from the spectrophotometer to the normalization system  107 . In another embodiment, the spectrophotometer may print the data to a screen or to a printer, and the data may be input into the normalization system  107  from, for example and without limitation, a keyboard or a scanner. 
     The normalization system  107  may receive the data from the spectrophotometer, and may calculate dilution data for all or a portion of the wells of the plate based at least in part on the data received from the spectrophotometer. The dilution data may indicate how much liquid to add to the wells to allow for a consistent concentration between the samples. The normalization system  107  may provide the dilution data to the liquid handling device  109 . The data may be provided by a network or a dedicated connection between the normalization system  107  and the liquid handling device  109 , or by a removable storage from the normalization system  107  to the liquid handing device. In another embodiment, the normalization system  107  may print the data to a screen or to a printer, and the data may be input into the liquid handing device from, for example and without limitation, a keyboard or a scanner. 
     The liquid handling device  109 , or liquid handler, may accept the plate, and may receive the dilution data from the normalization system  107 . The liquid handling device  109  may dispense liquid into the wells of the plate according to the dilution data provided by the normalization system  107 . The wells in the plate may, through the selective addition of additional liquid, be normalized across the plate or across one or more of the wells of the plate. The plate may then be removed from the normalization system  107 , shown in box  111 . 
       FIG. 2  shows a component view of the normalization system  107  of  FIG. 1  according to an embodiment of the present disclosure. The normalization system  107  may include an input module  203 , a calculation module  205 , and an output module  207 . The normalization system  107  may be a single system, or may be two or more systems in communication with each other. The normalization system  107  may include one or more input devices, one or more output devices, one or more processors, and memory associated with the one or more processors. The memory associated with the one or more processors may include, but is not limited to, memory associated with the execution of the modules, and memory associated with the storage of data. The normalization system  107  may also be associated with one or more networks, and may communicate with one or more additional systems via the one or more networks. The modules may be implemented in hardware or software, or a combination of hardware and software. The normalization system  107  may also include additional hardware and/or software to allow the normalization system  107  to access the input devices, the output devices, the processors, the memory, and the modules. The modules, or a combination of the modules, may be associated with a different processor and/or memory, for example on distinct systems, and the systems may be located separately from one another. In one embodiment, the modules may be executed on the same system as one or more processes or services. The modules may be operable to communicate with one another and to share information. Although the modules are described as separate and distinct from one another, the functions of two or more modules may instead be executed in the same process, or in the same system. 
     The input module  203  may receive data from an input device  201 . The input module  203  may also receive input over a network from another system. For example, and without limitation, the input module  203  may receive one or more signals from a computer over one or more networks. The input module  203  may receive data from the input device  201 , and may rearrange or reprocess the data so that it may be transmitted to the calculation module  205 . 
     The input device  201  may communicate with the input module  203  via a dedicated connection or any other type of connection. For example, and without limitation, the input device  201  may be in communication with the input module  203  via a Universal Serial Bus (“USB”) connection, via a serial or parallel connection to the input module  203 , or via an optical or radio link to the input module  203 . The transmission may also occur via one or more physical objects. For example, the spectrophotometer may generate one or more files, and may copy the one or more files to a removable storage device, such as a USB storage device or a hard drive, and a user may remove the removable storage device from the normalization system  107  and attach it to the input module  203  of the normalization system  107 . Any communications protocol may be used to communicate between the input device  201  and the input module  203 . For example, and without limitation, a USB protocol or a Bluetooth protocol may be used. 
     In one embodiment, the input device  201  may be a spectrophotometer. The spectrophotometer may analyze a plate and produce florescence data regarding one or more samples within wells on the plate. The data may be in the form of one or more files, or the spectrophotometer may print the data to a screen or a printer, and the data may be input into the normalization system  107  by, for example and without limitation, a keyboard, mouse, or scanner. 
     The network may include one or more of: a local area network, a wide area network, a radio network such as a radio network using an IEEE 802.11x communications protocol, a cable network, a fiber network or other optical network, a token ring network, or any other kind of packet-switched network may be used. The network may include the Internet, or may include any other type of public or private network. The use of the term “network” does not limit the network to a single style or type of network, or imply that one network is used. A combination of networks of any communications protocol or type may be used. For example, two or more packet-switched networks may be used, or a packet-switched network may be in communication with a radio network. 
     The calculation module  205  may receive inputs from the input module  203 , and may perform one or more calculations on the inputs. For example, and without limitation, the calculation module  205  may calculate the amount of liquid, if any, to be provided to each of the one or more wells on a plate. The calculation may be based on the florescence of each of the samples in the wells on the plate, or may be based on another calculated number from a spectrophotometer. For example, the spectrophotometer may calculate the concentration of the sample based on the florescence, and the calculation module  205  may use the concentration information to determine the amount of liquid, if any, to be provided to the well. One set of calculations may be based on the concentration calculated by the spectrophotometer. 
     For example, in one exemplary embodiment, a concentration of DNA of 5 ng/μL may be desired for the samples within each of the wells on a plate. If the concentration of a sample in a particular well is less than 5 ng/μL, then the sample may be properly diluted or overdiluted, and the system may not add additional liquid to the well containing the sample. The system may assign the value of liquid to be added to be zero microliters. If the concentration of the sample is greater than or equal to 200 ng/μL, liquid may be added to the maximum physical size of the well. The system may assign the value of liquid to be added to be 199 microliters, if the well on the plate can hold more than 200 microliters of liquid. If the concentration of the sample is between 5 and 200 ng/μL, the amount of liquid may be calculated according to the formula: (200*concentration in ng/μL)/5−200, to yield an amount of liquid to be added to the sample in microliters. Different calculations may be used if different concentrations are desired, or if the wells of the plate have a different total volume. The use of DNA is exemplary only, and other biomolecular components may be analyzed and/or processed. 
     Sample Visual Basic Code is as follows (code for user form): 
     
       
         
           
               
               
             
               
                   
                   
               
             
            
               
                   
                 Sub Norm _Macro( ) 
               
               
                   
                 ‘ 
               
               
                   
                 ‘ Norm _Macro Macro 
               
               
                   
                 ‘ Macro recorded 8/18/2009 by Annette R. Thom 
               
               
                   
                 ‘ 
               
               
                   
                 ‘ Keyboard Shortcut: Ctrl+f 
               
               
                   
                 ‘ 
               
               
                   
                 filetoopen = Application  —   
               
               
                   
                  .GetOpenFilename(“Exce 1 Files (*.xls), *.xls”) 
               
               
                   
                 If filetoopen &lt;&gt; False Then 
               
               
                   
                 Dim FileName As String 
               
               
                   
                 FileName = Left(filetoopen, Len(filetoopen) − 4) 
               
               
                   
                 FileName = FileName &amp; “.csv” 
               
               
                   
                 Workbooks.Open (filetoopen) 
               
               
                   
                 Sheets(“Sheet1 ”).Select Range(“B 1 7”).Select 
               
               
                   
                 Range(Selection, Selection.End(xlDown)). Select Selection.Copy 
               
               
                   
                 Sheets(“ Sheet2”).Select Range(“B 1 ”).Select 
               
               
                   
                 ActiveSheet.Paste 
               
               
                   
                 Sheets(“Sheet1 ”).Select ActiveWindow. SmallScroll Down:=−66 
               
               
                   
                 Range(“D1 7”). Select 
               
               
                   
                 Range(Selection, Selection.End(xlDown)). Select 
               
               
                   
                 Application.CutCopyMode = False Selection.Copy 
               
               
                   
                 Sheets(“ Sheet2”) .Select Range(“C1 ”).Select 
               
               
                   
                 ActiveSheet.Paste 
               
               
                   
                 Range(“A1 ”).Select 
               
               
                   
                 Application.CutCopyMode = False 
               
               
                   
                 ActiveCell.FormulaR1C1 = “Source ” 
               
               
                   
                 Range(“A2”). Select ActiveCell.FormulaR 1C1 = “1” 
               
               
                   
                 Selection.AutoFill Destination:=Range(“A2 :A93”) 
               
               
                   
                 Range(“A2 :A93 ”). Select 
               
               
                   
                 Range(“D 1 ”).Select 
               
               
                   
                 ActiveCell.FormulaR 1C1 = “H20” 
               
               
                   
                 Range(“D2”). Select 
               
               
                   
                  Application.Window State = xlNormal 
               
               
                   
                  ActiveCell.FormulaR1C1 =  —   
               
               
                   
                   “=IF(RC[− 1 ]&lt;5,0,IF(((200*RC[−1 ])/ 
               
               
                   
                   5)−200&gt;200,200,(200*RC[−1 ]/5)−200))” 
               
               
                   
                  Range(“D2”). Select 
               
               
                   
                  Selection.AutoFill Destination:=Range(“D2 :D93”) 
               
               
                   
                  Range(“D2:D93 ”). Select 
               
               
                   
                  Range(“D2”). Select 
               
               
                   
                  Application.DisplayAlerts = False Sheets(“ Sheet3 ”). Select 
               
               
                   
                  ActiveWindow.SelectedSheets.Delete Sheets(“Sheet1 ”).Select 
               
               
                   
                  ActiveWindow.SelectedSheets.Delete ActiveWorkbook. SaveAs 
               
               
                   
                  FileName, xl CSV 
               
               
                   
                  ActiveWorkbook.Close False 
               
               
                   
                  Application.DisplayAlerts = True 
               
               
                   
                  Application.Quit 
               
               
                   
                 End If 
               
               
                   
                 End Sub 
               
               
                   
                   
               
            
           
         
       
     
     The output module  207  may receive an input, and may transmit the input to an output device  209 . In one embodiment, the output module  207  may receive the input from the calculation module  205  in the form of alphanumeric data, and may transmit the data to the output device  209 . The output module  207  and the output device  209  may be in communication with one another. For example, and without limitation, the output module  207  and the output device  209  may be in communication via a network, or may be in communication via a dedicated connection, such as a cable or radio link. The output module  207  may also reformat the data received from the calculation module  205  into a format usable by the output device  209 . For example, the output module  207  may create one or more files that may be read by the output device  209 . In one embodiment, the output module  207  may reformat the data into one or more electronic files readable by Biomek® software, or other software suitable for creating or storing data. 
     The output device  209  may, in one embodiment, be a liquid handling device  109 . In one embodiment, the liquid handling device  109  may be a Biomek® NXp liquid handling device  109  or another device suitable for handling or delivering liquids. The output module  207  may communicate with the liquid handling device  109  by transmitting one or more electronic files to the liquid handling device  109 . The transmission may occur over a dedicated link, for example a USB connection or a serial connection, or may occur over one or more network connections. The transmission may also occur via one or more physical objects. For example, the output module  207  may generate one or more files, and may copy the one or more files to a removable storage device, such as a USB storage device or a hard drive, and a user may remove the removable storage device from the normalization system  107  and attach it to the liquid handing device. 
     Turning now to  FIG. 3 , a flowchart showing a method  300  of normalizing samples is shown according to an embodiment of the present disclosure. The method may begin in step  301 . As represented in step  303 , a plate may be prepared. The plate may contain one or more wells, and each of the wells may contain a different sample. The plate may be analyzed with a spectrophotometer  105 . The spectrophotometer  105  may be of any type, and may analyze the samples in the wells of the plate to determine florescence. The spectrophotometer  105  may analyze each sample individually by analyzing each of the one or more wells of the plate individually, or the spectrophotometer may analyze one or more samples simultaneously. The spectrophotometer  105  may record data regarding the sample, such as well location, florescence information, and data that may identify the plate uniquely or may uniquely identify the test of the plate or any of the samples in the wells on the plate. The spectrophotometer  105  may also perform one or more calculations to create additional data based on the florescence information. 
     As represented in step  305 , the input module  203  of the normalization system  107  may receive the data from the spectrophotometer  105 . The normalization system  107  may receive the data from the spectrophotometer  105  when the spectrophotometer  105  transmits the data to the normalization system  107 , such as over a network or other data transmission medium. The normalization system  107  and/or the input module  203  may reconfigure or rearrange the data received from the spectrophotometer  105  to perform additional calculations and transmit the data to the liquid handling device  109 . An exemplary set of data for a sample plate received from a spectrophotometer is shown in  FIG. 5 . In  FIG. 5 , the column “BackCalConc” may be used to calculate the additional liquid, if any, to be added to each well in the plate. 
     As represented in step  307 , the calculation module  205  of the normalization system  107  may calculate dilution data. The dilution data may be calculated based at least in part on the data received from the spectrophotometer  105 . For example, the florescence data from the spectrophotometer  105  for each of the wells on the plate may be used to calculate an additional amount of liquid to be added to the wells in order to normalize the concentration of DNA in each of the wells, or of one or more of the wells of the plate. 
     In the exemplary  96  well plate  401  shown in  FIG. 4 , samples in wells that are appropriately diluted may be represented by horizontal lines, represented by wells b 1 , b 5 , c 8 , c 9 , e 5 , e 6 , e 10 , e 12 , f 3 , f 6 , f 10 , g 6 , and h 12 . Samples in wells that receive 199 microliters of liquid are represented by vertical lines, represented by wells a 1 , a 3 , a 5 , a 6 , a 7 , a 9 , b 2 , b 6 , b 8 , b 10 , b 12 , c 1 , c 2 , c 3 , c 4 , c 5 , c 7 , d 1 , d 5 , d 8 , d 11 , e 2 , e 3 , e 7 , e 8 , e 9 , f 1 , f 4 , f 8 , g 2 , g 4 , g 8 , g 11 , h 2 , h 3 , h 5 , and h 9 . The remainder of the wells that have neither horizontal nor vertical markings may represent the wells that may receive a variable amount of liquid according to the calculations provided above with respect to the calculation module  205 . 
     The calculation module  205  may determine an amount of liquid to be added to each of the wells on the plate, and may output the dilution data to the output module  207 . The output module  207  may transmit the dilution data to the liquid handling device  109 , as represented in step  309 . Sample dilution data from the exemplary data received from the spectrophotometer  105  in  FIG. 5  is shown as  FIG. 6 . In  FIG. 6 , the well identification information and the “BackCalConc” data from  FIG. 5  is provided, as well as a determination of an amount of water to be added to each of the wells. The determination may be made by the calculation module  205  according to one or more formulas provided in the calculation module  205 . Additional exemplary variables for the calculation module may be shown in  FIG. 7 . The additional variables may allow a user to select one or more of the wells to calculate, or may allow the user to repeat the method for additional plates, so that many plates may be processed sequentially by the normalization system  107 . 
     As represented in step  311 , the liquid handling device  109  may receive the dilution data, and also the plate prepared as represented in step  703  and read by the spectrophotometer  105 . The dilution data may be transmitted from the output module  207  of the normalization system  107  by, for example and without limitation, a dedicated connection or a network, or through physical media. The liquid handling device  109  may read the dilution data, and may add an amount of liquid to each of the wells of the plate, or one or more wells of the plate, based on the values transmitted in the dilution data. The liquid handling device  109  may also be programmed with the maximum volume of the wells of the plate, or the liquid handling device  109  may determine the maximum volume of the wells of the plate by, for example and without limitation, determining the size of the plate or using a camera to determine the size of the plate or of the wells. The liquid handling device  109  may not add more liquid to a particular well than the well&#39;s maximum volume. The plate may then be removed from the liquid handling device  109 , and the normalized plate may then be analyzed further. The method may then end, as represented in step  713 . 
     While this disclosure has been described as having exemplary designs, the present disclosure can be further modified within the spirit and scope of this disclosure. This application is therefore intended to cover any variations, uses or adaptations of the disclosure using its general principles. Further, this application is intended to cover such departures from the present disclosure as come within known or customary practice in the art to which this disclosure pertains and which fall within the limits of the appended claims.