Allelic discrimination analysis using an efficiency related value (efr)

Abstract

In various embodiments this invention provides novel methods for discriminating two or more different target nucleic acids. In certain embodiments the methods comprise providing data amplification reactions comprising reagents to amplify two or more different target nucleic acids where the data comprise signals comprising an amplitude measurement representing the degree of amplification of each target nucleic acid in the amplification reaction and the time point in the amplification reaction at which the amplitude is measured; determining an efficiency related transform of the data, determining an efficiency related value for each target nucleic acid that is the maximum magnitude of the efficiency related transform; and outputting the efficiency related values in the amplification reaction for each target nucleic acid, where the relative amplitudes of the efficiency related values for each target nucleic acid is an indicator of the presence of each of said nucleic acids in said sample.

1 - 31 . (canceled) 
     
     
         32 . A machine-readable medium that provides instructions that, if executed by a machine, will cause the machine to perform operations comprising:
 receiving signals from one or more amplification reactions comprising reagents to amplify two or more different target nucleic acids from a single sample where the signals provide data comprising an amplitude measurement representing the degree of amplification of each target nucleic acid in the amplification reaction and the time point in the amplification reaction at which the amplitude is measured, and where the signal provides such data for a multiplicity of time points in the amplification reaction(s);   determining an efficiency related transform of said data where said efficiency related transform provides an amplitude measure that is related to the efficiency of amplification in said reaction;   determining an efficiency related value for each target nucleic acid that is the maximum magnitude of the efficiency related transform determined for that target nucleic acid; and   outputting to a display, printer, or storage medium the efficiency related values and corresponding points in the amplification reaction for each target nucleic acid, where the relative amplitudes of the efficiency related values for each target nucleic acid is an indicator of the presence of each of said nucleic acids in said sample.   
     
     
         33 . The medium of  claim 32 , wherein said data provide amplitude measurements for two or more target nucleic acids are in a single amplification reaction. 
     
     
         34 . The medium of  claim 32 , wherein said data provide amplitude measurements from a separate amplification reaction each reaction amplifying different target nucleic acids. 
     
     
         35 . The medium of  claim 32 , wherein said data provide amplitude measurements from a separate amplification reaction each reaction amplifying a single target nucleic acid. 
     
     
         36 . The medium of  claim 32 , wherein the time points in the amplification reaction are measured in cycle number. 
     
     
         37 . The medium of  claim 32 , wherein the points in the amplification reaction are measured in reaction time. 
     
     
         38 . The medium of  claim 32 , wherein said amplifying comprises amplifying at least three different target nucleic acids. 
     
     
         39 . The medium of  claim 32 , wherein said amplifying comprises amplifying at least five different target nucleic acids. 
     
     
         40 . The medium of  claim 32 , wherein said target nucleic acids comprise a nucleic acids derived from a first allele of a gene and a nucleic acid derived from a second allele of a said gene. 
     
     
         41 . The medium of  claim 40 , wherein outputting comprises outputting information indicating whether said sample is homozygous for said first allele, homozygous for said second allele or heterozygous for both alleles. 
     
     
         42 . The medium of  claim 32 , wherein the efficiency related transform is selected from the group consisting of the ratio transform of the signals, the shifted ratio transform of the signals, the first derivative of the signals, the differences between sequential signals, and the slope or gradient of the log of the signals. 
     
     
         43 . The medium of  claim 32 , wherein the efficiency related transform (ERT) is calculated as:
   ERT=(Signal n+1 /Signal n )−1 or  (a)
     ERT=(Signal n /Signal n−1 )−1  (b)
   
       where Signal n  is the signal produced at cycle number n, Signal n+1  is the signal produced at the subsequent cycle number, Signaln−1 is the signal produced at the previous cycle number, and n ranges from 1 up to the number of amplification cycles analyzed in the reaction for formula (a) and n ranges from 2 up to the number of amplification cycles −1 analyzed in the reaction for formula (b). 
     
     
         44 . The medium of  claim 32 , wherein the efficiency related value is the maximum of the efficiency related transform. 
     
     
         45 . The medium of  claim 32 , wherein the efficiency related value is the maximum gradient of the log of the amplification response. 
     
     
         46 . The medium of  claim 32 , wherein the efficiency related value is the maximum ratio of the amplification response. 
     
     
         47 . The medium of  claim 32 , wherein the efficiency related value is the maximum first derivative of the amplification response. 
     
     
         48 . The medium of  claim 32 , wherein additional signal values are generated by interpolating points between the measured signal values. 
     
     
         49 . The medium of  claim 48 , wherein said additional signal values are generated by interpolating points between the measured signal values using cubic splines. 
     
     
         50 . The medium of  claim 32 , wherein said efficiency related transform additionally provides a measure of the time or cycle number in said amplification reaction(s). 
     
     
         51 . The medium of  claim 32 , wherein said operations further comprise calculating a reaction point that is the fractional cycle number or time at which the maximum magnitude of the efficiency related transform occurs. 
     
     
         52 . The medium of  claim 32 , wherein said operations further comprise calculating an adjusted reaction point. 
     
     
         53 . The medium of  claim 52 , wherein the adjusted reaction point is equal to the reaction point minus the log base 2 of the efficiency related value. 
     
     
         54 . The medium of  claim 52 , wherein the adjusted reaction point is equal to the reaction point minus the log base 2 of the signal intensity above background. 
     
     
         55 . The medium of  claim 32 , wherein:
 said determining an efficiency related value for each target nucleic acid that is the maximum magnitude comprises identifying a peak in the efficiency related transform as a function of time or cycle number.   
     
     
         56 . The medium of  claim 55 , wherein said operations further comprise:
 determining the width of said peak;   comparing the width of the peak to a selected range of acceptable peak widths; and   outputting to a display, printer, or storage medium and indicator identifying the nucleic acid amplification reaction as possibly abnormal if the peak width determined is greater than or less than a selected range of acceptable peak widths.   
     
     
         57 . The medium of  claim 56 , wherein the peak width is calculated using only efficiency related transforms that occur at or before the reaction point value of the efficiency related value. 
     
     
         58 . The medium of  claim 32 , wherein amplification reaction is performed with a set of probes that comprises a FRET probe that is complementary to all or a portion of one of the amplified target nucleic acids. 
     
     
         59 . The medium of  claim 32 , wherein amplification reaction is performed with a set of probes that comprise a molecular beacon that is complementary to all or a portion of one of the amplified target nucleic acids. 
     
     
         60 . The medium of  claim 32 , wherein said receiving comprises receiving amplification data from an operator, or a computer readable medium. 
     
     
         61 . The medium of  claim 32 , wherein said providing comprises reading a data file from a PCR reaction. 
     
     
         62 . The medium of  claim 32 , wherein said receiving comprises receiving amplification data from a network. 
     
     
         63 . The medium of  claim 32 , wherein said receiving comprises receiving previously collected amplification data. 
     
     
         64 . The medium of  claim 32 , wherein said receiving comprises receiving previously amplification data as it is generated. 
     
     
         65 . The medium of  claim 32 , wherein said medium is a component of a nucleic acid amplification system. 
     
     
         66 . The medium of  claim 32 , wherein said medium is selected from the group consisting of a magnetic medium, a flash memory, an optical memory, a DRAM, and an SRAM. 
     
     
         67 . A system comprising:
 a device for performing a nucleic acid amplification and providing output signals that comprises a measure of the time point of the reaction, and the magnitude of the amplification of a target nucleic acid;   a processor operably coupled to said device; and   a machine-readable medium according to  claim 32 .   
     
     
         68 . The system of  claim 67 , wherein said processor also controls the amplification reaction(s) performed by said device. 
     
     
         69 . The system of  claim 67 , wherein said processor does not control the amplification reaction(s) performed by said device.