Method and an apparatus for determining nucleotide sequence, and a computer program product to be executed by the apparatus

Abstract

A method for determining nucleotide sequence encompasses: injecting a solution containing a sample DNA into a chip cartridge provided with a detecting electrode, to which a probe DNA is immobilized; introducing an intercalator solution in the chip cartridge; obtaining a current-voltage characteristic curve by measuring a current in the solution due to an electrochemical reaction of the intercalator through the detecting electrode; obtaining a baseline by linearly approximating the current-voltage characteristic curve; obtaining a net current value by subtracting, from a peak current value of the current-voltage characteristic curve, a baseline current value obtained from the baseline at a peak voltage value defining the peak current value; and identifying a nucleotide sequence in the sample DNA, using the net current value.

1 . A method for determining nucleotide sequence comprising: 
 injecting a solution containing a sample DNA into a chip cartridge provided with a detecting electrode, to which a probe DNA is immobilized;    introducing an intercalator solution in the chip cartridge;    obtaining a current-voltage characteristic curve by measuring a current in the solution due to an electrochemical reaction of the intercalator through the detecting electrode;    obtaining a baseline by linearly approximating the current-voltage characteristic curve;    obtaining a net current value by subtracting, from a peak current value of the current-voltage characteristic curve, a baseline current value obtained from the baseline at a peak voltage value defining the peak current value; and    identifying a nucleotide sequence in the sample DNA, using the net current value.    
   
   
       2 . The method of  claim 1 , before obtaining the baseline by linearly approximating the current-voltage characteristic curve, further comprising: 
 obtaining a tail line by linearly approximating the current-voltage characteristic curve, in a voltage range between a predetermined lower limit voltage and a predetermined higher limit voltage;    calculating a slope of the tail line;    determining whether the current-voltage characteristic curve is a normal profile or an abnormal profile, using the slope of the tail line; and    excluding the current-voltage characteristic curve which is assigned as the abnormal profile.    
   
   
       3 . The method of  claim 2 , wherein determining whether the current-voltage characteristic curve is the normal profile or abnormal profile comprises: 
 determining whether the slope of the tail line lie in a slope range between a predetermined lower limit slope value and a predetermined higher limit slope value; and    assigning the normal profile to the current-voltage characteristic curve having the slope of the tail line lying in the slope range, and the abnormal profile to the current-voltage characteristic curve having the slope of the tail line lying out of the slope range.    
   
   
       4 . The method of  claim 1 , wherein obtaining the net current value comprises: 
 obtaining a differential curve of the current-voltage characteristic curve, with respect to voltage;    defining the peak voltage value as a voltage value at which the differential curve zero-crosses in a voltage range between predetermined lower and upper limit values;    defining the peak current value obtained from the current-voltage characteristic curve at the peak voltage value;    defining an inflection point at which the differential curve is minimized in a voltage range which is less than the peak voltage value;    determining a linear expression passing through the peak current point and the inflection point;    obtaining an intersection-point voltage value at an intersection-point of the current-voltage characteristic curve and the linear expression;    defining an offset voltage by subtracting a predetermined offset value from the intersection-point voltage value;    obtaining the baseline by linearly approximating the current-voltage characteristic curve between the offset voltage and the intersection-point voltage;    obtaining the baseline current value, by assigning the peak voltage value for the baseline; and    obtaining the net current value by subtracting from the peak current value, a baseline current value.    
   
   
       5 . The method of  claim 1 , when DNA chip cartridge have a plurality of the detecting electrodes, a data set including a plurality of data of the net current values for each detecting electrode, respectively, is obtained, prior to obtaining the baseline current value, further comprising: 
 excluding abnormal data from the data set.    
   
   
       6 . The method of  claim 5 , wherein excluding the abnormal data comprises: 
 dividing the data set into a plurality of groups so that each of the group implements data set defined for a unit combination of equivalent detecting electrodes; and    eliminating data with abnormal value under certain criterion from every group, by repeating a sequence of process-steps to eliminate data with abnormal value from an original data set of one of the groups until all of the abnormal data are eliminated from all of the groups.    
   
   
       7 . The method of  claim 6 , wherein the elimination of data with abnormal value from the original dataset of one of the groups comprises: 
 calculating a standard deviation and a mean-value of the original data set of the group;    calculating a first CV value, by dividing the standard deviation by the mean-value;    comparing the first CV value with a predetermined standard CV value; and    determining all net current values are normal in the original dataset of the group, when the first CV value is smaller than the predetermined standard CV value, and determining an abnormal value is included in the original data set of the group when the first CV value is equal to or larger than the predetermined standard CV value.    
   
   
       8 . The method of  claim 7 , wherein, in a case when the first CV value is equal to or larger than the predetermined standard CV value, elimination of data with abnormal value from the original dataset of one of the groups comprises: 
 preliminarily eliminating the data with a minimum value from the original dataset of the group so as to define a tentative dataset of the group;    calculating standard deviation and mean-value of the net current values of a tentative dataset of the group;    calculating a second CV value of the tentative dataset, through dividing the standard deviation of the tentative dataset by the mean-value of the tentative dataset;    comparing the second CV value with the first CV value multiplied by a predetermined CV value correction coefficient;    finally eliminating the data with the minimum value from the original dataset so as to define a new dataset, when the second CV value is smaller than the first CV value multiplied by the predetermined CV value correction coefficient, 
 calculating a new first CV value of the new dataset, through dividing the standard deviation of the new dataset by the mean-value of the new dataset, comparing the new first CV value with the predetermined standard CV value,  
 determining all net current values are normal in the new dataset, when the new first CV value is smaller than the predetermined standard CV value, and  
 preliminarily eliminating data with the minimum value from the new dataset when the new first CV value is equal to or larger than the predetermined standard CV value then returning to the calculation of the second CV value; and  
   returning to the original dataset so as to preliminarily eliminate data with a maximum value from the original dataset so as to define a second tentative dataset, and calculating a third CV value of the second tentative dataset, through dividing standard deviation of the second tentative dataset by mean-value of the second tentative dataset, when the second CV value is equal to or larger than the first CV value multiplied by the predetermined CV value correction coefficient.    
   
   
       9 . The method of  claim 8 , wherein, in a case when the second CV value is equal to or larger than the first CV value multiplied by the predetermined CV value correction coefficient, the elimination of the abnormal data comprises: 
 finally eliminating the data of the maximum value from the original dataset so as to define a second new dataset, when the third CV value is smaller than the first CV value multiplied by the predetermined CV value correction coefficient;    calculating another new first CV value of the second new dataset, through dividing the standard deviation of the second new dataset by the mean-value of the second new dataset, so as to compare the new first CV value with the predetermined standard CV value;    determining all net current values are normal in the second new dataset, when the new first CV value is smaller than the predetermined standard CV value; and    preliminarily eliminating data with a minimum value from the second new dataset when the new first CV value is equal to or larger than the predetermined standard CV value so as to return to the calculation of the third CV value.    
   
   
       10 . A method for determining nucleotide sequence comprising: 
 injecting a sample DNA into a chip cartridge having: 
 a plurality of detecting electrodes, on which a probe DNA is immobilized, and  
 a control electrode, on which a DNA which has nucleotide sequence different from the first and second probe DNA is immobilized, or a DNA is not immobilized;  
   obtaining detection signals through the detecting electrodes and control signals through the control electrodes;    calculating a mean-value of the detection signals;    calculating a mean-value of the control signals;    comparing a difference derived by subtracting the mean-value of the control signals from the mean-value of the detection signals with a predetermined signal-increment criterion; and    determining the presence of the sample DNA.    
   
   
       11 . The method of  claim 10 , further comprising: 
 calculating a first standard deviation of the detection signals;    calculating a second standard deviation of the control signals; and    comparing a ratio of the difference to a sum of the first and the second standard deviations with a predetermined effective scale factor so as to judge reliability of the determination of the presence of the sample DNA.    
   
   
       12 . A method for determining nucleotide sequence comprising: 
 injecting a sample DNA into a chip cartridge comprising: 
 a plurality of first detecting electrodes, on which a first probe DNA is immobilized,  
 a plurality of second detecting electrodes, on which a second probe DNA, which has nucleotide sequence different from the first probe DNAs is immobilized, and  
 a plurality of control electrodes, on which a control DNA, which has nucleotide sequence different from the first and second probe DNA is immobilized, or a DNA is not immobilized;  
   obtaining first detection signals through first detecting electrodes, second detection signals through second detecting electrodes, and control signals through control electrodes;    calculating mean-values of the first detection signals, the second detection signals, and the control signals, respectively;    comparing a first mean-value difference derived by subtracting the mean-value of the control signals from the mean-value of the first detection signals with a predetermined signal-increment criterion, and comparing a second mean-value difference derived by subtracting the mean-value of the control signals from the mean-value of the second detection signals with the predetermined signal-increment criterion; and    proceeding to a procedure for genotyping the sample DNA, when at least one of the first and the second mean-value differences is equal to or larger than the predetermined signal-increment criterion, and determining that the genotyping is not possible when both the first and the second mean-value differences is smaller than the predetermined signal-increment criterion.    
   
   
       13 . The method of  claim 12 , wherein a hetero type is identified for the sample DNA, when both of the first and the second mean-value differences are equal to or larger than the predetermined signal-increment criterion.  
   
   
       14 . The method of  claim 13 , further comprising: 
 calculating a first standard deviation of the first detection signals, a second standard deviation of the second detection signals, and a third standard deviation of the control signals;    comparing a ratio of the first mean-value difference to a sum of the first and the third standard deviation with a predetermined effective scale factor; and    comparing a ratio of the second mean-value difference to a sum of the second and the third standard deviation with the predetermined effective scale factor so as to judge reliability of the identification of the hetero type.    
   
   
       15 . The method of  claim 12 , wherein, when both of the first and the second mean-value differences are equal to or larger than the predetermined signal-increment criterion, the procedure for genotyping the sample DNA comprises: 
 comparing a logarithm of the absolute value of a ratio of the first mean-value difference to the second mean-value difference with a first homo-typing criterion, a second homo-typing criterion, and a hetero-typing criterion, so as to identify a first homo-type, a second homo-type, or a hetero-type, respectively.    
   
   
       16 . The method of  claim 15 , further comprising: 
 calculating a first standard deviation of the first detection signals, a second standard deviation of the second detection signals, and a third of the control signals standard deviation;    comparing a ratio of the first mean-value difference to a sum of the first and the third standard deviations with a predetermined effective scale factor; and    comparing a ratio of the second mean-value difference to a sum of the second and the third standard deviations with the predetermined effective scale factor so as to judge reliability of the identification of the first homo-type, the second homo-type, or the hetero-type.    
   
   
       17 . The method of  claim 12 , wherein, when only the first mean-value difference is equal to or larger than the predetermined signal-increment criterion, the procedure for genotyping the sample DNA comprises: 
 comparing a mean-value ratio of the mean-value of the first detection signals to the mean-value of the first control signals with a predetermined significant scale-factor; and    identifying a first homo-type when the mean-value ratio is equal to or larger than the significant scale-factor.    
   
   
       18 . The method of  claim 17 , further comprising: 
 calculating a first standard deviation of the first detection signals and a second standard deviation of the first control signals; and    comparing a ratio of the first mean-value difference to a sum of the first and the second standard deviations with a predetermined effective scale factor so as to judge reliability of the identification of the first homo-type.    
   
   
       19 . The method of  claim 12 , wherein, when only the first mean-value difference is equal to or larger than the predetermined signal-increment criterion, the procedure for genotyping the sample DNA comprises: 
 comparing a mean-value ratio of the mean-value of the first detection signals to the mean-value of the second detection signals with a predetermined significant scale-factor; and    identifying a first homo-type when the mean-value ratio is equal to or larger than the significant scale-factor.    
   
   
       20 . The method of  claim 19 , further comprising: 
 calculating a first standard deviation of the first detection signals and a second standard deviation of the second detection signals; and    comparing a ratio of the first mean-value difference to a sum of the first and the second standard deviations with a predetermined effective scale factor so as to judge reliability of the identification of the first homo-type.    
   
   
       21 . An apparatus for determining nucleotide sequence comprising: 
 a chip cartridge having: 
 a plurality of first detecting electrodes on which a first probe DNA is immobilized,  
 a plurality of second detecting electrodes, on which a second probe DNA which has nucleotide sequence different from the first probe DNAs is immobilized, and  
 a plurality of control electrodes, on which a control DNA, which has nucleotide sequence different from the first and second probe DNA is immobilized, or a DNA is not immobilized;  
   a detecting system for measuring currents through the first detecting electrodes, the second detecting electrodes, and the control electrodes;    a current-profile judgement module configured to acquire first detection signals from the first detecting electrodes, second detection signals from the second detecting electrodes, and control signals from the control electrodes, as current-voltage characteristic curves through the detecting system, to obtain slopes of tail lines in each of the current-voltage characteristic curves, to assign normality or abnormality of the current-voltage characteristics curve from the slopes of the tail lines, and to exclude abnormal detection signals from calculation object; and    a net current calculation module configured to subtract a baseline current value from the current-voltage characteristic curves measured by the first detection signals, the second detection signals, and the control signals from corresponding peak currents in the current-voltage characteristic curves measured by the first detection signals, the second detection signals, and the control signals, respectively, so as to obtain net currents for the first detection signals, the second detection signals, and the control signals.    
   
   
       22 . The apparatus of  claim 21 , further comprising: 
 a temperature controller configured to control temperature of the chip cartridge;    a fluid transport system configured to transport a solution into the chip cartridge; and    a control mechanism configured to control the detecting system, temperature controller, and the fluid transport system.    
   
   
       23 . The apparatus of  claim 21 , wherein the net current detection module comprises: 
 a voltage calculation unit configured to the current-voltage characteristic curves with respect to voltage value so as to obtain differential curves, and to obtain peak voltage values where the differential curve zero crosses;    a baseline approximation unit configured to obtain linear expressions, which linearly approximate the current-voltage characteristic curves; and    a net-current-value calculation unit configured to subtract baseline current values, which are obtained by assigning the peak voltage values for the linear expression, from peak current values at the peak voltage values so as to obtain net peak current values in the current-voltage characteristic curves.    
   
   
       24 . The apparatus of  claim 21 , further comprising: 
 an abnormal-data eliminating module configured to eliminate abnormal data from dataset assigned to a group, which is implemented by data of a plurality of net current values calculated by the net current calculation module;    a presence judgement module configured to determine whether target nucleic acid is present or not, using the dataset of the net current values, from which abnormal data have been eliminated; and    a typing module configured to genotype sample DNA.    
   
   
       25 . A computer program product to be executed by an apparatus for determining nucleotide sequence, the computer program product comprising: 
 instructions configured to obtain first detection signals through first detecting electrodes, second detection signals through second detecting electrodes, and control signals through control electrodes, after injecting a sample DNA into a chip cartridge comprising: 
 a plurality of first detecting electrodes on which a first probe DNA is immobilized,  
 a plurality of second detecting electrodes, on which a second probe DNA, which has nucleotide sequence different from the first probe DNAs is immobilized, and  
 a plurality of control electrodes, on which a control DNA, which has nucleotide sequence different from the first and second probe DNA is immobilized, or a DNA is not immobilized;  
   instructions configured to calculate mean-values of the first detection signals, the second detection signals, and the control signals, respectively; and    instructions configured to compare a first mean-value difference derived by subtracting the mean-value of the control signals from the mean-value of the first detection signals with a predetermined signal-increment criterion, and comparing a second mean-value difference derived by subtracting the mean-value of the control signals from the mean-value of the second detection signals with the predetermined signal-increment criterion,    wherein the apparatus proceeds to a procedure for genotyping the sample DNA, when at least one of the first and the second mean-value differences is equal to or larger than the signal-increment criterion, and determine that the genotyping is not possible when both the first and the second mean-value differences is smaller than the signal-increment criterion.