Spectral calibration of fluorescent polynucleotide separation apparatus

Abstract

The invention relates to methods, compositions, and systems for calibrating a fluorescent polynucleotide separation apparatus. One aspect of the invention is multiple color calibration standards and their use. A multiple color calibration standard is a mixture of at least two polynucleotides of different length, wherein each of the polynucleotides is labeled with a spectrally distinct fluorescent dye. Another aspect of the invention is to produce total emission temporal profiles of multiple color calibration standards for use in calibrating fluorescent polynucleotide separation apparatus. The peaks corresponding to the fluorescently labeled polynucleotides in the total emission temporal profile may be detected using a peak detector that is driven by changes in the slopes of the total emission temporal profile. Calibration of fluorescent polynucleotide separation apparatus, with various embodiments of the methods of the invention, includes the step of identification of the labeled polynucleotides of the multiple color calibration standards. The process of spectral calibration of a fluorescent polynucleotide separation apparatus using a multiple color calibration standard may include the step of the estimating (extracting) of the dyes' reference spectra, using information from the peak detection process performed on the total emission temporal profile. Other aspects of the invention include systems for separating and detecting fluorescently labeled polynucleotides, wherein the system is designed for spectral calibration in accordance with the subject calibration methods employing multiple color calibration standards. Another aspect of the invention is methods and compositions for detecting the flow of electrical current through a separation channel of a fluorescent polynucleotide separation apparatus. These methods and compositions employ monitoring dyes. Monitoring dyes are fluorescent dyes that are spectrally distinct from the dye on the polynucleotide intended to convey genetic information, e.g., fluorescent polynucleotide sequencing reaction products.

1 . A method of monitoring a separation channel of a fluorescent polynucleotide separation apparatus, said method comprising: 
 (i) introducing a fluorescently labeled polynucleotide composition to an inlet end of a separation channel of a fluorescent polynucleotide separation apparatus, said composition comprising (a) a polynucleotide labeled with a first fluorescent dye, and (b) a monitoring dye that is spectrally distinct from the first fluorescent dye;    (ii) causing the fluorescently labeled polynucleotide composition to migrate down the channel; and    (iii) detecting for the monitoring dye at one or more regions downstream of said inlet end;    whereby detection of the monitoring dye at said one or more regions is indicative of flow along the channel.    
     
     
         2 . The method of  claim 1 , wherein the fluorescently labeled polynucleotide composition comprises: 
 a plurality of polynucleotides comprising at least two polynucleotides labeled with at least two respective spectrally distinct fluorescent dyes, wherein the monitoring dye is spectrally distinct from each of the at least two spectrally distinct fluorescent dyes.    
     
     
         3 . The method of  claim 2 , wherein the plurality of polynucleotides labeled with at least two spectrally distinct fluorescent dyes comprises a polynucleotide sequencing reaction product mixture.  
     
     
         4 . The method of  claim 1 , wherein the monitoring dye is attached to a polynucleotide.  
     
     
         5 . The method of  claim 1 , wherein the monitoring dye produces significant emission when excited by the same excitation source or sources used to excite other fluorescent dyes of the fluorescently labeled polynucleotide composition.  
     
     
         6 . The method of  claim 1 , further comprising detecting current in the separation channel by detecting migration of the monitoring dye.  
     
     
         7 . The method of  claim 1 , further comprising detecting separation of the sequencing reaction products by detecting migration of the monitoring dye.  
     
     
         8 . A method of monitoring a separation channel of a fluorescent polynucleotide separation apparatus, said method comprising: 
 (i) introducing a fluorescently labeled polynucleotide composition to an inlet end of a separation channel of a fluorescent polynucleotide separation apparatus, said composition comprising (a) at least two spectrally distinct fluorescent dyes, wherein each of the at least two dyes is correlated with a different polynucleotide base, and (b) a monitoring dye that is spectrally distinct from each of the at least two spectrally distinct fluorescent dyes;    (ii) causing the fluorescently labeled polynucleotide composition to migrate down the channel; and    (iii) detecting for the monitoring dye at one or more regions downstream of said inlet end;    whereby detection of the monitoring dye at said one or more regions is indicative of flow along the channel.    
     
     
         9 . The method of  claim 8 , wherein the fluorescently labeled polynucleotide composition comprises a polynucleotide sequencing reaction product mixture.  
     
     
         10 . The method of  claim 8 , wherein the monitoring dye is attached to a polynucleotide.  
     
     
         11 . The method of  claim 8 , wherein the monitoring dye produces significant emission when excited by the same excitation source or sources used to excite the at least two spectrally distinct fluorescent dyes of the fluorescently labeled polynucleotide composition.  
     
     
         12 . The method of  claim 8 , further comprising detecting current in the separation channel by detecting migration of the monitoring dye.  
     
     
         13 . The method of  claim 8 , further comprising detecting separation of the sequencing reaction products by detecting migration of the monitoring dye.  
     
     
         14 . The method of  claim 8 , wherein the at least two spectrally distinct fluorescent dyes comprise four spectrally distinct fluorescent dyes.  
     
     
         15 . The method of  claim 8 , wherein the at least two spectrally distinct fluorescent dyes comprise at least four spectrally distinct fluorescent dyes.  
     
     
         16 . A computer readable medium having computer code for detecting a monitoring dye at one or more regions downstream of an inlet end of a separation channel of a fluorescent polynucleotide separation apparatus, during a method comprising: 
 (i) introducing a fluorescently labeled polynucleotide composition to the inlet end, the composition comprising (a) at least two spectrally distinct fluorescent dyes, wherein each of the at least two dyes is correlated with a different polynucleotide base, and (b) the monitoring dye, the monitoring dye being spectrally distinct from each of the at least two spectrally distinct fluorescent dyes; and    (ii) causing the fluorescently labeled polynucleotide composition to migrate down the channel,    wherein detection of the monitoring dye at the one or more regions is indicative of flow along the channel.    
     
     
         17 . The computer readable medium of  claim 16 , further comprising computer code for detecting current in the separation channel by detecting migration of the monitoring dye.  
     
     
         18 . The computer readable medium of  claim 16 , further comprising computer code for detecting separation of sequencing reaction products by detecting migration of the monitoring dye.  
     
     
         19 . The computer readable medium of  claim 16 , further comprising: 
 computer code for causing a computer to compare a signal-to-noise ratio of a first dye to a minimum signal-to-noise ratio; and    computer code for causing a computer to determine whether the signal-to-noise ratio of the first dye exceeds the minimum signal-to-noise ratio.    
     
     
         20 . An electronic computer comprising the computer readable medium of  claim 16  and programmed with the computer code.