Abstract:
A method for designing a primer for real-time PCR comprises retrieving a primer set using a known primer search algorithm and based on primer design conditions, visually displaying the retrieved primer set together with its splicing information and base sequence, narrowing the primer set using narrowing conditions, and visually displaying a narrowed primer set together with its splicing information and base sequence.

Description:
BACKGROUND OF THE INVENTION 
   1. Field of the Invention 
   The present invention relates to a method for designing a primer in a real-time PCR (to be hereafter referred to as RT-PCR). 
   2. Background Art 
   As one of the means for comparing the amount of expression of genes, a method whereby the amount of mRNA accumulated in cells is determined is widely employed. RT-PCR is widely utilized in recent years as a method for measuring the amount of accumulated mRNA. In a general procedure involving RT-PCR, the accumulated amount of mRNA is measured as follows. Samples grown under different conditions, or samples of different series grown under identical conditions are prepared, and mRNA is extracted from a tissue of concern. Using the thus extracted mRNA as a template, PCR is conducted under conditions including a primer set specific to a target gene and a fluorescent dye that binds to double strands to emit color. The fluorescent intensity in a reaction solution is measured in parallel with the reaction, whereby the number of nucleic acids in the double strands is measured. Based on the increase in fluorescent intensity, the amount of amplification product is determined. 
   PCR is a highly sensitive process and is theoretically capable of performing amplification even from a few copies of template. If genomic DNA is mixed in the extracted mRNA, amplification even occurs based on the genomic DNA as a template. In this case, PCR products would include both those based on mRNA as a template and those based on genomic DNA as a template. As a result, the amount of mRNA would not be accurately measured. 
   With reference to  FIG. 1 , the relationship between the types of template and the length of PCR products is described. In a eukaryotic experimental system, when splicing a target gene, individual primers are designed on different exons. Genomic DNA  101  has intron  102  and exon  103 . Primer  104  on the sense strand side is represented by an arrow pointing to the right, and primer on the nonsense strand side by an arrow pointing to the left. When genomic DNA  101  is used as a template, a PCR product  106  would include an intron portion. When mRNA  110  is used as a template, a PCR product  111  would not include an intron portion and would therefore be shorter than when genomic DNA is used as a template. Based on the lengths of the PCR products, amplification products based on genomic DNA as a template can be distinguished from amplification products based on mRNA as a template. For this purpose, a method is required for designing a primer on exons with an intron portion disposed between them. 
   Furthermore, when the measured mRNA amount is compared between different samples, an analytical curve must be prepared. Theoretically, if the temperature cycle in PCR differs by one cycle, the amount of template doubles. However, in practice, this varies from one experiment to another, depending on experimental conditions, target base sequence, and primer sequence. Thus, for comparing the mRNA amount between different samples, a PCR product is prepared that includes only the target base sequence of the primer, and then an analytical curve is prepared using the dilution series of DNA that contains only the target base sequence as a template. Accordingly, a method is required for designing a primer for preparing a template that only contains the target base sequence of the primer for measuring the mRNA amount.
     Patent Document 1: JP Patent Publication (Kokai) No. 2001-245697 A   

   SUMMARY OF THE INVENTION 
   In experiment utilizing RT-PCR, an experiment-supporting functionality is required for designing a primer at positions with intron disposed between them. To realize such functionality, the following three conditions must be met:
     (1) The genomic base sequence and mRNA base sequence of a target gene have been determined by prior studies.   (2) The primer can be designed at positions that do not include a splicing site on exons sandwiching an intron with an experimentally measurable length.   (3) The information about the designed primer can be displayed in an easy-to-understand manner for the preparation of an analytical curve and for determination purposes, thereby facilitating comparison and analysis.   

   It is therefore an object of the invention to provide a method whereby the aforementioned conditions  2  and  3  can be satisfied and whereby a primer for real-time PCR can be easily designed. 
   The invention provides a method for designing a primer for real-time PCR, comprising the steps of: 
   entering the base sequence data for a target gene; 
   determining whether or not said base sequence data contains splicing information; 
   acquiring splicing information if it is determined in said determination step that splicing information is not contained; 
   entering primer design conditions for the designing of a primer at positions of exon with intron disposed between them; 
   retrieving a primer set using a known primer search algorithm and in accordance with said primer design conditions; 
   visually displaying a retrieved primer set, together with said splicing information and said base sequence; 
   entering a condition for narrowing the displayed primer set; and 
   visually displaying said primer set narrowed using the narrowing condition, together with said splicing information and base sequence. 

   
     BRIEF DESCRIPTION OF THE DRAWINGS 
       FIG. 1  illustrates the relationship between types of templates and the length of PCR products. 
       FIG. 2  shows a block diagram of a RT-PCR primer designing apparatus of the invention. 
       FIG. 3  shows an example of a screen for the entry of data. 
       FIG. 4  shows an example of a screen for the entry of primer designing parameters. 
       FIG. 5  shows a screen for narrowing display results. 
       FIG. 6  shows a screen for displaying measurement primer results. 
       FIG. 7  shows the result of designing a primer for preparing a template for creating an analytical curve. 
       FIG. 8  shows a flowchart outlining the processes performed in accordance with the invention. 
       FIG. 9  shows a flowchart of the processes for calculating splicing information. 
       FIG. 10  shows a flowchart of the processes for retrieving a sequence suitable for a primer from a target region. 
   

   DESCRIPTION OF THE PREFERRED EMBODIMENTS 
   An embodiment of the invention is described in the following with reference to the drawings.  FIG. 2  shows a block diagram of a RT-PCR primer designing apparatus according to the invention. The RT-PCR primer designing apparatus of the present embodiment includes a file  200  containing the base sequence data about a target gene and splicing information; a parameter input screen for primer designing purposes; a display unit  201  for displaying the information about the primer that has been designed; input means  202  including a keyboard and mouse; a file  203  containing the designed-primer information; a central processing unit  204 ; and a program memory  205  in which various programs are stored. 
   The program memory  205  stores search programs  206  and drawing programs  207 . The search programs  206  include a splicing information calculation program  208  for calculating splicing information, and a primer sequence search program  209  for a primer designing process, namely, for retrieving a primer sequence. The drawing programs  207  include a file selection program  210  for displaying a screen for the entry of a file to be selected as a template; a parameter input program  211  for displaying a screen for the entry of primer designing parameters (conditions); a re-search screen display program  212  for displaying a screen on which primer search results can be narrowed; and a detailed information display program  213  for displaying a detailed information screen for an mRNA amount measuring primer. The primer designing process is carried out in the wizard format. When any of these programs is run, a dialog-format screen is displayed. When a process is initiated by the primer designing apparatus of the present embodiment, the file selection program  210  is initially run, whereby a screen shown in  FIG. 3  is displayed. 
     FIG. 3  shows an example of the file-selection screen that is displayed when the file selection program  210  is run. The screen incorporates a radio button  300  for selecting a file with annotation information as a template, and another radio button  301  for selecting a file without annotation information as a template. As an example of the file with annotation information, a published gene database file can be mentioned. As an example of the file without annotation information, a genomic DNA and mRNA base sequence data prepared by the user can be mentioned. The user selects either one of the radio buttons. The selected field is placed in an input-activated state. The field that has not been selected is placed in an input-deactivated state. 
   Selecting the button  300  activates a button  303  for displaying a list of files in the published gene database for clicking. When the button  303  is clicked and a file is selected, a path of the selected file is displayed in a text box  302 . 
   Selecting the button  301  activates a button  305  for displaying a list of the files of the genomic DNA base sequence, and a button  307  for displaying a list of the mRNA base sequence files, for clicking. When the button  305  or  307  is clicked and a file is selected, the path of the selected file is displayed in a text box  304  or  306 . Alternatively, the user may directly enter a path in the text box  302 ,  304 , or  306 . 
   When the process is to be terminated, a cancel button  308  is depressed. The process can be continued by depressing a button  309 . 
   If the button  309  is depressed when the button  300  has been selected, the parameter input program  211  is activated, whereby the screen shown in  FIG. 4  is displayed. If the button  309  is depressed when the button  301  has been selected, the splicing information calculation program  208  is activated, whereby the splicing information is acquired. After the splicing information is thus acquired, the parameter program  211  is run, whereby the screen shown in  FIG. 4  is displayed. 
   In the above example, it is assumed that the files in the published gene database contain splicing information while the genomic DNA and mRNA base sequence data prepared by the user do not contain splicing information. However, there are cases in which published gene databases do not contain splicing information and the splicing information is obtained from the genomic DNA sequence data and mRNA base sequence data prepared by the user. 
     FIG. 4  shows an example of a screen for the entry of primer designing conditions (parameters), which is displayed when the parameter input program  211  is run. The screen includes a field  400  for displaying splicing information; a field  410  for the entry of primer search conditions; a field  420  for the entry of PCR product conditions; and a field  430  for designating design options. The field  400  shows a gene name  401  and splicing information in a schematic fashion. The splicing information includes schematic representations of mRNA base sequence  402 , full length  403 , intron number  404 , exon number  405 , the length of intron sequence removed by splicing  406 , and position  407  of binding by splicing. 
   The field  410  includes the length and value width  411  of a designed primer; Tm value  412 , GC content  413 , and a check box  414  for designating an option for selecting only those primer candidates whose  3 ′ terminal is G or C. The field  420  includes the length and value width  421  of PCR product obtained by the designed primer; a text box  422  for the entry of a minimum value of the difference in length of PCR products [The length of PCR product when genomic DNA is used as template—the length of PCR product when mRNA is used as template]; and a text box  423  for the entry of a minimum value of intron ratio [The length of intron/the length of PCR product when mRNA is used as template× 100 ]. The user makes an entry in either one of the two text boxes  422  and  423 . 
   The field  430  includes a check box  431  for designating the exon number on the left and right sides for designing each primer; and a check box  432  for designating the intron number displaced between exons. The two check boxes  431  and  432  may be selected simultaneously. When a plurality of exon numbers or intron numbers are entered, a comma “,” may be inserted between the individual numbers. The screen further includes a button  441  for returning to the previous data input screen shown in  FIG. 3 , and a button  442  for proceeding to the next process. When the button  442  is depressed, the primer sequence search program  209  is initiated. When the primer sequence search program  209  is being run, a screen shown in  FIG. 5  is displayed. 
     FIG. 5  shows an example of the screen that is displayed when the re-search screen display program  212  is being run, for the entry of conditions for narrowing the search results. The screen includes a text box  500  for displaying the total number of primer sets that have been retrieved; a text box  501  for displaying the total number of retrieved primers on the sense strand side; a text box  502  for displaying the total number of retrieved primers on the nonsense strand side; a field  503  for the entry of parameters for narrowing the displayed results; and a check box  504  for designating an option for displaying only those primers among the primer combinations that most well correspond to conditions. The field  503  includes individual areas for the Tm value, GC content, and the length of primer. 
   The screen further includes a refresh button  505 , a button  506  for returning to the dialog screen for designating the primer designing conditions shown in  FIG. 4 ; and a button  507  for proceeding to the next process. If the refresh button  505  is depressed after narrowing conditions have been entered in the field  503 , only those primer information is extracted that correspond to the conditions, and the information is reflected on the values in the text boxes  500 ,  501 , and  502 . When the button  507  is clicked, the detailed information display program  213  is initiated, and a screen shown in  FIG. 6  is displayed. 
     FIG. 6  shows an example of the screen for displaying the detailed results of designing an mRNA amount-measuring primer, which is displayed when the detailed information display program  213  is run. This screen includes a field  600  for displaying primer information in a schematic fashion, and a field  610  for displaying the detailed information about the primer set selected in the field  600 . The field  600  includes, in addition to the field  601  for displaying the splicing information about the target gene, a field  602  for displaying the position information regarding the primer set. With regard to the primer set, a primer set herein refers to a plurality of primers on the nonsense strand side relative to one primer on sense strand side. If the position information regarding a primer set cannot be displayed all at once, the display area is moved using a scroll bar  603 . 
   When a line corresponding to a particular primer set is selected in the field  602 , the detailed information about that primer set is displayed in the field  610 . The field  610  shows the primer name; primer base sequence; position on the mRNA base sequence where complementary binding of the 5′ terminal of primer occurs; primer length; Tm and GC contents of primer; and the length of PCR product in a case where mRNA is used as template and a case where genomic DNA is used as template. 
   The field  610  includes a check box  613  for selecting all of the primer sets and a check box  614  for selecting a single primer set. The screen also includes a button  615  for checking all of the primer sets; a button  616  for proceeding to the primer design step for preparing a template for creating an analytical curve; a button  617  for returning to the previous process; and a button  618  for ending the primer designing and outputting the information about a selected primer to the file  203 . 
   The button  616  can be clicked when the check box  614  is selected. Specifically, a set of primers is selected when preparing a template for the creation of an analytical curve. When the button  616  is clicked, a condition excluding an area sandwiched by a set of primers selected in the check box  614  is added in the parameters for primer design, and then primer designing starts. Thereafter the screen shown in  FIG. 5  for narrowing the display results is displayed again, and the above-described procedure is performed. When the button  507  in the screen of  FIG. 5  is depressed, a screen for displaying the result of the primer for preparing an analytical curve-creating template is displayed, as shown in  FIG. 7 . 
     FIG. 7  shows an example of the screen for displaying the result of designing a primer for preparing an analytical curve-creating template. The screen includes a field  700  for displaying primer information in a schematic fashion, and a field  710  for displaying the detailed information about the primer set selected in the field  700 . In the present example, the field  701  for displaying the splicing information shows the position information  703  for the measurement primer set selected in the check box  614  in the screen shown in  FIG. 6 . The screen also includes a button  713  for checking all of the primer sets; a button  714  for returning to the previous process; and a button  715  for ending the primer designing and adding the information about the selected primer to the file  203 . When a line corresponding to a set of primers is selected in the field  702  and then the button  715  is depressed, the result of primer designing for preparing the selected template for analytical curve creating purposes is added to the selected measurement primer information. The screen then returns to the screen shown in  FIG. 6 . 
     FIG. 8  shows an outline of the processes performed by the primer designing apparatus of the invention. At step S 801 , base sequence data is entered. At step S 802 , it is determined whether the entered base sequence data contains splicing information. As described above, when the radio button  300  for selecting the use of a file in a published gene database as a template of  FIG. 3  has been clicked, the base sequence data includes splicing information, so that the routine proceeds to step S 804 . On the other hand, when the radio button  301  has been clicked to select the use of the base sequence data for genomic DNA and mRNA prepared by the user as a template of  FIG. 3 , the base sequence data does not contain splicing information, so that the routine proceeds to step S 803 . At step S 803 , a process for calculating the splicing information is performed, of which the details will be described with reference to  FIG. 9 . At step S 804 , the screen of  FIG. 4  is displayed, which includes the field  400  for displaying the splicing information, and fields  410 ,  420 , and  430  for the entry of primer design conditions (parameters). At step S 805 , a primer design process is carried out based on the thus entered parameters, of which the details will be described with reference to  FIG. 10 . At step S 806 , it is determined whether or not the primer designing for preparing an analytical curve-creating template is to be conducted. If the primer designing for preparing an analytical curve-creating template is to be conducted, the area sandwiched by the measurement primer set selected in the check box  614  in the screen of  FIG. 6  is excluded from the processed object, and the primer designing process is performed again. At step S 808 , information about the primer that has been designed is outputted to the file  203 , and the routine ends. 
     FIG. 9  shows an outline of the process for calculating the splicing information at step S 803 . The process is carried out by the pricing information calculating process program  208 . At step S 901 , genomic DNA and mRNA base sequence data are entered. At step S 902 , the base sequence is processed as a character string, and an initial value of zero is substituted in the variable “DNA position” and the variable “mRNA position.” An initial value of zero is also entered in the variable “exon number,” which counts the number of exons. At step S 903 ,  10  characters from the “mRNA position” in the mRNA base sequence are substituted in a character string “Searched sequence.” At step S 904 , sequences corresponding to the genomic DNA base sequence are retrieved from the “DNA position.” The mRNA base sequences obtained by splicing are all included in the genomic DNA base sequence. At step S 905 , it is determined if there is a corresponding sequence. If not, the input data is erroneous, and therefore the process terminates. If there is a corresponding sequence, the subscript of the corresponding letter in the genomic DNA base sequence is used as the corresponding position, and is substituted into the variables “exon start position” and “DNA position” at step S 906 . Because there is exon in this case, the “exon number” is incremented. At step S 907 , the “mRNA position” and “DNA position” are incremented. 
   Steps S 908  to S 910  constitute a search loop, in which the letter following the “DNA position” in the genomic DNA base sequence and the letter in the “mRNA position” in the mRNA base sequence are compared. If they correspond, the “mRNA position” and “DNA position” are incremented at step S 909 , and then it is determined once again if they correspond. The comparison is repeated until they do not correspond. When they cease to correspond, this means the end of exon, and therefore the routine advances to step S 911 . 
   At step S 911 , the “DNA position” is substituted into the variable “exon end position,” and the “exon number,” “exon start position,” and “exon end position” are added to the splicing information. 
   At step S 912 , it is determined whether the “mRNA position” is at the end of the mRNA sequence. If the “mRNA position” is not at the end of the mRNA sequence, the routine returns to step S 903 . If the “mRNA position” is at the end of the mRNA sequence, the routine advances to step S 913  where the splicing information is outputted. 
     FIG. 10  shows an outline of the process for retrieving a sequence from a target area at step S 805  that is suitable for a primer. This process is carried out by the primer sequence search program  209 . At step S 1001 , the primer designing conditions (parameters) entered in the fields  410 ,  420 , and  430  of  FIG. 4  are acquired. At step S 1002 , it is determined if the parameter values are correct. If they are not correct, the routine advances to step S 1003  where a re-entry is prompted. If the parameters are correct, they are delivered to the existing primer search algorithm at step S 1004 , and a process is carried out to retrieve a sequence suitable as a primer. At step S 1005 , based on the search result, primers on the nonsense string side with corresponding positional relationship condition relative to a primer on the sense string side are retrieved and grouped into a primer set. At step S 1006 , the obtained primer information is displayed in the field  602  shown in  FIG. 6 . At step S 1007 , it is determined whether or not the displayed information is to be narrowed. Specifically, it is determined whether the refresh button  505  of  FIG. 5  has been clicked. If the information is to be narrowed, primer information corresponding to the conditions entered in the field  503  of  FIG. 5  is retrieved at step S 1008 . At step S 1009 , the detailed information about the corresponding primer set is displayed in the field  602  of  FIG. 6 . At step S 1010 , the information about the primer set selected by the user is acquired, which completes the entire process. 
   While a preferred embodiment of the invention has been described using specific terms, such description is for illustrative purposes only, and it is to be understood that changes and variation may be made without departing from the spirit or scope of the following claims.