Real time gene expression profiling
View Patent ↗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. A method for monitoring the amplification of a set of nucleic acid sequences of interest, said 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; and one oligonucleotide in each said pair of oligonucleotides is detectably labeled;
b) subjecting the mixture resulting from step (a) to an amplification regimen comprising at least two iterative cycles of nucleic acid strand separation, oligonucleotide primer annealing and polymerase extension of annealed primers, wherein during the amplification regimen, following at least one 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 detectable label in a distinctly sized amplification product present in said aliquot;
d) for each distinctly sized amplification product detected in step (c), calculating a cycle number, C t , where the amount of that amplification product crosses a predefined threshold, and correlating the threshold cycle with the amount of a nucleic acid having a sequence of interest in said sample, wherein said method provides an amplification profile and a relative abundance for members of said set of nucleic acid sequences of interest.
2. The method of claim 1 wherein said detectable label comprises a fluorescent dye.
3. The method of claim 1 wherein aliquot removal of step (b) is performed after each cycle in said amplification regimen.
4. The method of claim 1 wherein said separating nucleic acid molecules comprises capillary electrophoresis.