Multiplex Q-PCR Arrays

Abstract

This invention provides methods and systems for measuring the concentration of multiple nucleic acid sequences in a sample. The nucleic acid sequences in the sample are simultaneously amplified, for example, using polymerase chain reaction (PCR) in the presence of an array of nucleic acid probes. The amount of amplicon corresponding to the multiple nucleic acid sequences can be measured in real-time during or after each cycle using a real-time microarray. The measured amount of amplicon produced can be used to determine the original amount of the nucleic acid sequences in the sample.

What is claimed is: 
     
         1 . A method comprising: (a) performing a nucleic acid amplification on two or more nucleotide sequences to produce two or more amplicons in a fluid wherein the array comprises a solid surface with a plurality of nucleic acid probes at independently addressable locations; and (b) measuring the hybridization of the amplicons to the two or more nucleic acid probes while the fluid is in contact with the array to obtain an amplicon hybridization measurement. 
     
     
         2 . The method of  claim 1  further comprising using the amplicon hybridization measurement to determine the concentration of the amplicons in the fluid. 
     
     
         3 . The method of  claim 2  further comprising using the amplicon hybridization measurement to determine the original amount of nucleotide sequences. 
     
     
         4 . The method of  claim 3  wherein the concentration of amplicon is measured during or after some, but not all of the amplification cycles. 
     
     
         5 . The method of  claim 3  wherein the concentration of amplicon is measured during or after each of the amplification cycles. 
     
     
         6 . The method of  claim 3  wherein the concentration of amplicon is measured during on average every 2, 3, 4, 5, 6, 7, 8, 9 or 10 amplification cycles. 
     
     
         7 . The method of  claim 3  wherein the nucleic acid amplification is polymerase chain reaction (PCR), and an amplification cycle corresponds with a temperature cycle. 
     
     
         8 . The method of  claim 7  wherein at least some temperature cycles comprise (a) a probe hybridization phase at one temperature, and (b) primer annealing phase at a higher temperature than the probe hybridization temperature. 
     
     
         9 . The method of  claim 7  wherein at least some temperature cycles comprise 4 or more temperature phases, wherein one or more of the phases is a probe hybridization phase wherein the hybridization of amplicons to the nucleic acid probes is measured. 
     
     
         10 . The method of  claim 9  wherein the temperature is changed during probe hybridization phase. 
     
     
         11 . The method of  claim 9  comprising two or more hybridization phases, carried out at different temperatures. 
     
     
         12 . The method of  claim 7  wherein at least some temperature cycles comprise a first denaturing phase, a probe hybridization phase, a second denaturing phase, a primer annealing phase, and an extension phase. 
     
     
         13 . The method of  claim 1  wherein the array is in contact with the fluid during the amplification. 
     
     
         14 . The method of  claim 1  wherein the two or more nucleotide sequences are not only two complementary nucleotide sequences. 
     
     
         15 . The method of  claim 1  wherein the amplification is an isothermal amplification. 
     
     
         16 . The method of  claim 1  wherein the amplification is a linear amplification. 
     
     
         17 . The method of  claim 1  wherein the measuring of hybridization comprises measuring the kinetics of hybridization of the amplicons to the nucleic acid probes. 
     
     
         18 . The method of  claim 15  wherein the measuring of the kinetics of hybridization comprises measuring a light signal at multiple time points. 
     
     
         19 . The method of  claim 1  wherein the amplicons comprise a quencher. 
     
     
         20 . The method of  claim 19  wherein primers are used to create the amplicons and