Methods for quantitative amplification and detection over a wide dynamic range

A method for detecting a target nucleic acid sequence in a sample comprising:

• (a) providing a sample suspected of containing a target nucleic acid;
• (b) generating from the target nucleic acid a defined ratio of at least two differentiable amplicon species,
• wherein the at least two differentiable amplicon species are generated using a single amplification oligomer that hybridizes to one strand of the target nucleic acid and at least two amplification oligomers that hybridize to the complementary strand of the target nucleic acid,
• wherein the at least two amplification oligomers hybridizing to the complementary strand of the target nucleic acid are provided in different amounts and have distinct nucleic acid sequences, such that the defined ratio of the at least two differentiable amplicon species is generated and whereby the at least two differentiable amplicon species differ in nucleic acid composition;
• and
• (c) detecting the presence and amount of each generated amplicon species,
• wherein a first amplicon species is detectable in a first linear range representing a first concentration of the target nucleic acid in the sample to a second concentration of the target nucleic acid in the sample, and a second amplicon species is detectable in a second linear range representing a third concentration of the target nucleic acid in the sample to a fourth concentration of the target nucleic acid in the sample, and
• wherein the first concentration is less than the third concentration, which is less than the second concentration, which is less than the fourth concentration, such that said first and second linear ranges overlap and provide an extended dynamic range for determining the presence and amount of the target nucleic acid in the sample;
• wherein the at least two amplification oligomers hybridizing to the complementary strand of the target nucleic acid comprise identical nucleotide sequences except that each of the at least two amplification oligomers comprises a unique unhybridized nucleotide sequence that is at least one nucleobase in length and that is joined to the 5′ end of the target hybridizing region of the amplification oligomer;
• wherein the generating and detecting steps are each performed in a single vessel;
• and
• wherein the detecting step comprises hybridizing the at least two differentiable amplicon species with distinguishable probes that each hybridize to only one of the at least two differentiable amplicons.