Methods of analyzing chromosomal translocations using fluorescence in situ hybridization (fish)

Abstract

Probes and methods of using the probes to detect chromosomal rearrangements and/or deletions are provided. The methods utilize probes that are free of repeat sequences to provide greater selectivity and sensitivity; methods for producing such probes are also disclosed. The probe sets utilized in the detection methods are designed to hybridize to chromosomes at regions outside known breakpoints, instead of spanning the breakpoint as with conventional FISH methods, and, in some instances, are further designed to bind to regions located outside the genes involved in the rearrangement. Methods utilizing probe sets with two and four colors are also described, as are automated methods for analyzing rearrangements.

1 . A method for determining the presence or absence of a reciprocal chromosomal translocation involving a first gene, which includes potential breakpoints for the translocation, and a second gene, which includes potential breakpoints for the translocation, the first and second genes being located on first and second chromosomes, respectively, the method comprising: 
 (a) contacting, under hybridization conditions, a chromosomal preparation with a set of probes comprising first and second nucleic acid probes; wherein said chromosomal preparation comprises the first and second chromosomes; wherein the first probe bears a first label and is hybridizable to an untranslocated form of the first chromosome at a region located outside the first gene and no further than 1 Mb from the outermost known centromeric breakpoint associated with the translocation; wherein the second probe bears a second label different from the first label and is hybridizable to an untranslocated form of the second chromosome at a region located outside the second gene and no further than 1 Mb from the outermost known telomeric breakpoint associated with the translocation; wherein 
 (i) if the first and second chromosomes have undergone the reciprocal chromosomal translocation, the first and second probes hybridize to a derivative chromosome formed via the translocation, such that both the first and second labels appear on the derivative chromosome; whereas  
 (ii) if the first and second chromosomes have not undergone the reciprocal chromosomal translocation, the first and second probes hybridize to their respective chromosomes, such that only the first label appears on the first chromosome and only the second label appears on the second chromosome;  
   (b) detecting a pattern of hybridization for the first and second probes;    (c) determining from the pattern whether the first and second probes appear on the derivative chromosome or appear separately on the first and second chromosomes, respectively, thereby determining the presence or absence of the reciprocal chromosomal translocation.    
     
     
         2 . The method of  claim 1 , wherein the regions of chromosomal DNA bound by the first and second probes are 100-300 kb in length.  
     
     
         3 . The method of  claim 1 , wherein the regions of chromosomal DNA bound by the first and second probes are 10-1000 kb in length.  
     
     
         4 . The method of  claim 1 , wherein the region that the first probe hybridizes to is at least 400 kb and no further than 1 Mb from the outermost known centromeric breakpoint associated with the translocation and the region that the second probe hybridizes to is at least 400 kb and no further than 1 Mb from the outermost known telomeric breakpoint associated with the translocation, such that if the first and second chromosomes have undergone the translocation, the first and second probes as hybridized to the derivative chromosome are separated by at least 0.8 Mb.  
     
     
         5 . The method of  claim 1 , wherein the reciprocal chromosomal translocation is selected from the group consisting of: 
 t(11;14)(q13; q32) (BCL1; IGH);    t(14;18)(q32; q21) (IGH;BCL2);    t(8;14)(q24; q32) (MYC;IGH); and    t(9;22)(q34; q11) (BCR;ABL).    
     
     
         6 . The method of  claim 5 , wherein the reciprocal chromosomal translocation is t(11;14)(q13; q32) (BCL1; IGH).  
     
     
         7 . The method of  claim 5 , wherein the reciprocal chromosomal translocation is t(14;18)(q32; q21) (IGH;BCL2).  
     
     
         8 . The method of  claim 5 , wherein the reciprocal chromosomal translocation is t(8;14)(q24; q32) (MYC;IGH).  
     
     
         9 . The method of  claim 5 , wherein the reciprocal chromosomal translocation is t(9;22)(q34; q11) (BCR;ABL).