Homogeneous multiplex hybridization analysis by color and Tm

Abstract

The invention provides methods and devices for analyzing sequence variations in nucleic acid samples comprising multiple loci, each having two, three or more possible allelic sequences. The method involves combining at least a first and second pair of oligonucleotide probes with the nucleic acid sample. The first pair of probes is capable of hybridizing in proximity to each other within a segment of the nucleic acid sample comprising the first locus and the second pair is capable of hybridizing in proximity to each other within a segment of the nucleic acid sample comprising the second locus. The first member of each probe pair comprises a FRET donor and the second member comprises a FRET acceptor, the FRET acceptor of the first probe pair member having a different emission spectrum from the FRET acceptor of the second probe pair. Upon hybridization, the proximity of the first and second member of each probe pair is sufficient to allow fluorescence resonance energy transfer between the FRET donor and the FRET acceptor.

1. A method for analyzing a nucleic acid sample comprising three or more loci each having at least two different allelic sequences, said method comprising:
 (a) combining at least a first, a second and a third pair of oligonucleotide probes with said nucleic acid, each of the members of said pairs being capable of hybridizing in proximity to each other to a segment of said nucleic acid comprising at least one of said three or more loci, wherein (i) the first member of each pair comprises a FRET donor and the second member comprises a FRET acceptor, wherein the FRET acceptor of the second member in said first pair has an emission spectrum which is different from the emission spectrum of the FRET acceptor of said second and third oligonucleotide probe pairs, (ii) when said second and third probe pairs have the same FRET acceptor, each of said second and third probe pairs has a different Tm from each other for each different allele within the nucleic acid segment to which each member hybridizes (iii) upon hybridization, the proximity of the members of a probe pair is sufficient to allow fluorescence resonance energy transfer between said FRET donor and said FRET acceptor, and (iv) at least one of said members of each pair has a sequence which results in the differential hybridization of that member with at least two different alleles which may be present at said loci;  
 (b) measuring the emission of each of said FRET acceptors at a first temperature; and  
 (c) repeating said emission measurements at a second temperature; wherein the emission of said FRET acceptors at different temperatures provides an indication of the alleles present at said three or more loci.  
 
     
     
       2. The method of  claim 1 , wherein the FRET acceptor of each of the second members of each of a first, a second and a third probe pair has an emission spectrum which is different from the emission spectrum of the others. 
     
     
       3. The method of  claim 1  or  2 , wherein said nucleic acid sample is the product of one or more reactions selected from the group consisting of PCR, 3SR, SDA and RCA. 
     
     
       4. The method of  claim 1  or  2 , wherein at least one probe comprises two FRET acceptors, two FRET donors or a FRET acceptor and a FRET donor, and said probe is a member of two different probe pairs. 
     
     
       5. The method of  claim 1  or  2 , wherein steps (b) and (c) are repeated throughout a range of temperatures. 
     
     
       6. The method of  claim 5 , wherein said range is from at least 20° C. to at most 95° C. 
     
     
       7. The method of  claim 5 , wherein said emission measurements are repeated at least every 0.1 to 10 seconds. 
     
     
       8. The method of  claim 7 , wherein the temperature is varied at least 0.01 to 1° C. per second. 
     
     
       9. The method of  claim 1  or  2 , wherein said emission measurements at a particular temperature are simultaneous. 
     
     
       10. The method of  claim 1  or  2 , wherein at least one of said FRET acceptors is selected from the group consisting of LC Red 640, Cy 5, Cy 5.5 and LC Red 705. 
     
     
       11. The method of  claim 1  or  2  wherein said emission measurements are corrected for spectral overlap between or among the different fluorophores. 
     
     
       12. A method for analyzing a nucleic acid sample comprising multiple loci, comprising:
 ( a )  combining at least first, second and third oligonucleotide probes with said nucleic acid sample, wherein said second probe comprises two FRET acceptors, two FRET donors or a FRET acceptor and a FRET donor and is a member of first and second different probe pairs, where said first pair comprises said first and said second probes that hybridize in proximity to each other to a segment of said nucleic acid sample comprising a first locus and said second pair comprises said second and said third probes that hybridize in proximity to each other to a segment of a second locus, wherein said first and said second probes contain either a donor or acceptor so as to form donor - acceptor pairs upon hybridization of said first, second and third probes so that fluorescence resonance energy transfer between said FRET donor and said FRET acceptor occurs, where at least one of said members of said first pair has a sequence which results in the differential hybridization of that member with at least two different alleles which may be present at said first locus, and at least one of said members of said second pair has a sequence which results in the differential hybridization of that member with at least three different alleles which may be present at said second locus;      ( b )  measuring the emission of each of said FRET acceptors at a first temperature; and      ( c )  repeating said emission measurements at a second and third temperature;      
       
         wherein the emission of said FRET acceptors at different temperatures provides an indication of the alleles present at said first and second loci.