Multiple mode multiplex reaction quenching method
OwnerTony Godfrey, William McMillaninventors
Life of this patent
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Abstract
Methods for balancing multiplexed PCR reactions are provided which exploit differences in primer and amplicon Tms. The methods may be controlled by a computer process. Also provided are articles of manufacture useful in such methods and compositions containing primers and probes useful in such methods.
Claims21
A multiplexed nucleic acid amplification reaction method comprising amplifying in a reaction mix in two or more amplification stages a first nucleic acid sequence to produce a first amplicon and a second nucleic acid to produce a second amplicon, each amplification stage comprising one or more PCR cycles, each PCR cycle comprising a denaturing step, an annealing step and an elongation step that may be conducted at the same temperature as the annealing step, the first amplicon having a first Tm and the second amplicon having a second Tm that is different from the first Tm, wherein two or more of the amplification stages have different amplicon denaturation conditions, such that relative accumulation of the first amplicon and the second amplicon is modulated.
A method of identifying expression of markers indicative of the presence of breast cancer cells in a lymph node of a patient, comprising quantifying expression levels of TACSTD1, PIP and an endogenous control mRNA in a nucleic acid sample from a patient's lymph nodes.
A kit comprising:
- (a) packaging material;
- (b) a vessel contained within the packaging material containing a first primer pair and a second primer pair, wherein in a multiplexed PCR assay, the first PCR primer pair generates an amplicon having a first Tm and the second PCR primer pair generates an amplicon having a second Tm, different from the first;
- and
- (c) instructions describing a multiplexed nucleic acid amplification method comprising amplifying in a reaction mix a first nucleic acid sequence to produce a first amplicon and a second nucleic acid to produce a second amplicon, the first amplicon having a first Tm and the second amplicon having a second Tm that is different from the first Tm, wherein the reaction comprises two stages comprising two protocols with different amplicon denaturation conditions, such that relative accumulation of the first amplicon and the second amplicon is modulated.
Dependencies are inferred from the claim text.