Real time gene expression profiling

Abstract

The invention relates to methods of monitoring the amplification of one or more nucleic acid sequences of interest. More particularly, the invention relates to methods of monitoring the amplification of sequences of interest in real time. The methods disclosed herein provide methods for monitoring the amplification of one sequence or two or more sequences from a single sample, as well as methods for monitoring the amplification of one or more than one sequence from two or more samples. The monitoring methods of the invention permit improved determination of the abundance of one or more target nucleic acids, especially target RNA species, in one or more original samples.

1 . (canceled) 
     
     
         2 . A method for determining the threshold cycle, C t , for members of a set of nucleic acid sequences of interest in an amplification reaction, the method comprising:
 (a) contacting a nucleic acid sample in a reaction vessel with a set of pairs of oligonucleotide primers, wherein:   each said pair comprises a first oligonucleotide primer that specifically hybridizes with a nucleic acid molecule comprising said nucleic acid sequence of interest, and a second oligonucleotide primer that specifically hybridizes with a sequence comprised by the complement of said nucleic acid sequence of interest, wherein the primer extension product of one oligonucleotide primer, when separated from its complement, can serve as a template for the synthesis of the extension product of the other primer; each said pair of oligonucleotides is specific for one nucleic acid sequence of interest; each oligonucleotide primer pair in said set is selected so that it generates a distinctly sized amplification product in a subsequent amplification regimen;   b) subjecting the mixture resulting from step (a) to an amplification regimen comprising a plurality of iterative cycles of nucleic acid strand separation, oligonucleotide primer annealing and polymerase extension of annealed primers in the presence of a fluorescent label which is incorporated into products generated in said amplification regimen, wherein during the amplification regimen, following each of a plurality of said iterative cycles, an aliquot of said mixture is removed from said reaction vessel and nucleic acid molecules in said aliquot are separated;   c) for each aliquot removed and subjected to separation of nucleic acid molecules according to step (b), detecting the incorporation of fluorescent label in a distinctly sized amplification product present in said aliquot;   d) for each distinctly sized amplification product detected in step (c), determining a cycle number, C t , where the amount of that amplification product crosses a predefined detection threshold, such that a value for Ct is determined for each of said set of nucleic acid sequences of interest in said single amplification reaction.   
     
     
         3 . The method of  claim 1 , wherein the step of separating nucleic acid molecules in said aliquot is performed in real time during the amplification regimen. 
     
     
         4 . The method of  claim 1  wherein said fluorescent label is attached to said first or said second oligonucleotide primer of each pair of oligonucleotide primers. 
     
     
         5 . The method of  claim 1  wherein aliquot removal of step (b) is performed after each cycle in said amplification regimen. 
     
     
         6 . The method of  claim 1  wherein said separating nucleic acid molecules comprises capillary electrophoresis. 
     
     
         7 . The method of  claim 1  wherein aliquot removal and nucleic acid separation are performed by an automated routine in a modular apparatus comprising a thermal cycler, a sampling device and a capillary electrophoresis device. 
     
     
         8 . The method of  claim 1 , which permits Ct determination in a single amplification reaction for a set of nucleic acids of interest in which members of said set have different amplification efficiencies. 
     
     
         9 . The method of  claim 1 , further comprising the step, after aliquot removal, of replenishing the reaction mixture with a volume of reactant mixture equal to the amount removed.