Method for identifying or detecting genomic rearrangements in a biological sample

Abstract

A method for detection, visualization and/or comparison of polynucleotide sequences of interest using specially designed sets of long and short probes that enhance resolution and simplify visualization and detection. Probe compositions useful for practicing this method and procedures for identifying useful probes and probe combinations. These methods are useful for the detection of genomic rearrangements, especially those associated with various diseases, disorders and conditions including cancer or for assessment of genomic rearrangements associated with therapy. The probe compositions may be used in kits for detection of genetic rearrangements or in companion diagnostic products or kits, such as kits for the diagnosis or assessment of predisposition to cancer such as colorectal cancer.

1 - 48 . (canceled) 
     
     
         49 . A kit comprising a set of short probes or a set of short and a set of long probe(s); and optionally one or more components for binding said probes to a polynucleotide, for performing molecular combing, and/or for detecting whether hybridization has occurred; wherein said short probes 10 kb or less and said long probes are 12 kb or more; and
 (i) wherein the short probes comprise a set of probes that taken together bind to a continuous stretch of more than 12 kb of the genomic region of interest; or   (ii) wherein the long probes bind to sequences outside the genomic region of interest and do not overlap the short probe sequences;   and optionally, where the repetitive sequences have been removed from the long and/or short probes.   
     
     
         50 . A kit according to  claim 49  for the detection of genomic rearrangements associated with a condition selected from the group consisting of: colorectal cancer or genetic predisposition to colorectal cancer, breast cancer or genetic predisposition to breast cancer, ovarian cancer or genetic predisposition to ovarian cancer, and lung cancer or genetic predisposition to lung cancer. 
     
     
         51 . A composition containing a set of short, or short and long probe(s), wherein at least two of said probes detect a genetic rearrangement by using Molecular Combing, said short probes binding to at least one region of interest without gaps longer than 15 kb between the portions of the target sequence bound by the short probes in each region of interest and said composition comprising either
 at least one short probe of less than 10 kb and at least one non-overlapping long probe of more than 14 kb that binds to a sequence near but outside of the region(s) of interest; or   at least one group of at least two short probes, less than 10 kb each, which total length is longer than 14 kb and less than 150 kb, hybridizing contiguously on the genetic target.   
     
     
         52 . The composition of  claim 51 , wherein the short probe(s) range from 0.5 kb to 9 kb. 
     
     
         53 . The composition according to  claim 51 , wherein the long probe(s) range from 14 kb to 40 kb. 
     
     
         54 . The composition according to  claim 51 , wherein the size of the short probes range from 0.5 to 9 kb and wherein at least 90% of the frequent repetitive sequences have been removed from the short probes. 
     
     
         55 . The composition of according to  claim 51 , wherein the probe sequences hybridize specifically on the MSH2 gene or in the region of the MSH2 gene or on the MLH1 gene or in the region of the MLH1 gene. 
     
     
         56 . The composition according to  claim 51 , wherein said short probe sequence(s) are selected from the group consisting of the group of short probes obtained by amplification using the primer pairs disclosed as SEQ ID NO: 21-60, SEQ ID NO:95-122; SEQ ID NO:163-172; SEQ ID NO:185-202 and SEQ ID NO:227-248 or the long probe sequence(s) are selected from the group consisting of the group of long probe obtained by amplification using the primer pairs disclosed as SEQ ID NO: 61-76 and SEQ ID NO:123-138. 
     
     
         57 . A kit according to  claim 49  wherein the short probes are at least 500 bp each. 
     
     
         58 . A kit according to  claim 57  wherein the long probes are 14 kb or more, and optionally wherein the long probes are shorter than 150 kb. 
     
     
         59 . A kit according to  claim 58  for the detection of genomic rearrangements associated with a condition selected from the group consisting of: colorectal cancer or genetic predisposition to colorectal cancer, breast cancer or genetic predisposition to breast cancer, ovarian cancer or genetic predisposition to ovarian cancer, and lung cancer or genetic predisposition to lung cancer. 
     
     
         60 . A kit according to  claim 59 , wherein sequences of more than 200 bp, of which more than 10 copies with less than 20% mismatch are found within the regions of interest, have been removed from the short and/or long probes. 
     
     
         61 . A kit according to  claim 58 , wherein sequences of more than 200 bp, of which more than 10 copies with less than 20% mismatch are found within the regions of interest, have been removed from the short and/or long probes 
     
     
         62 . A kit according to  claim 59 , wherein sequences of more than 200 bp, of which more than 10 copies with less than 20% mismatch are found within the regions of interest, have been removed from the short and/or long probes 
     
     
         63 . A composition according to  claim 51 , wherein sequences of more than 200 bp, of which more than 10 copies with less than 20% mismatch are found within the regions of interest, have been removed from the short and/or long probes. 
     
     
         64 . A composition according to  claim 52 , wherein sequences of more than 200 bp, of which more than 10 copies with less than 20% mismatch are found within the regions of interest, have been removed from the short and/or long probes. 
     
     
         65 . A composition according to  claim 53 , wherein sequences of more than 200 bp, of which more than 10 copies with less than 20% mismatch are found within the regions of interest, have been removed from the short and/or long probes. 
     
     
         66 . A composition according to  claim 52 , wherein the sizes of long probes range from 14 kb to 150 kb, and wherein sequences of more than 200 bp, of which more than 10 copies with less than 20% mismatch are found within the regions of interest, have been removed from the short and/or long probes.