Compositions and methods for the detection of DNA topoisomerase II complexes with DNA

Abstract

Compositions, methods, and kits for detecting DNA topoisomerase II-DNA complexes are disclosed.

1 . A method for identifying sequences present in DNA topoisomerase H-DNA complexes in cells, comprising: 
 a) providing cells suspected of containing DNA topoisomerase H-DNA complexes;    b) isolating DNA topoisomerase H-DNA complexes from the cell;    c) amplifying the DNA present in said isolated DNA topoisomerase H-DNA complexes via polymerase chain reaction; and    d) identifying the sequences present in said amplified DNA, thereby identifying the sequences present in said DNA topoisomerase II-DNA complexes.    
     
     
         2 . The method of  claim 1 , wherein the DNA in said DNA topoisomerase 11-DNA complexes is genomic DNA.  
     
     
         3 . The method of  claim 1 , wherein the identification of the sequences in step d) comprises further amplifying the amplified DNA from step c) with gene specific primers.  
     
     
         4 . The method of  claim 1 , wherein the identification of the sequences in step d) comprises further amplifying the amplified DNA from step c) by real-time PCR.  
     
     
         5 . The method of  claim 1 , wherein the identification of the sequences in step d) comprises hybridizing the amplified DNA from step c) with a microarray.  
     
     
         6 . The method of  claim 1 , wherein said cells are CD34+ cells.  
     
     
         7 . The method of  claim 1 , wherein said amplified DNA of step c) comprises sequences from the MLL gene.  
     
     
         8 . The method of  claim 1 , wherein said cells are exposed to an agent suspected of modulating formation of topoisomerase cleavage complexes.  
     
     
         9 . The method of  claim 5 , wherein said microarray comprises MLL bcr oligonucleotide sequences.  
     
     
         10 . The method of  claim 9 , wherein said MLL bcr oligonucleotide sequences hybridize to non-repetitive MLL bcr sequences.  
     
     
         11 . The method of  claim 9 , wherein said microarray further comprises oligonucleotide sequences from the Alu region between nucleotide positions 663-1779 in the MLL bcr.  
     
     
         12 . The method of  claim 9 , wherein said microarray further comprises control sequences which are not involved in MLL translocations.  
     
     
         13 . The method of  claim 13 , wherein said control sequences are selected from the group consisting of MLL exon 25, MLL exon 3, GAPDH, c-myc, and bacterial gene sequences.  
     
     
         14 . The method of  claim 9 , wherein said microarray further comprises oligonucleotide sequences from MLL partner genes.  
     
     
         15 . The method of  claim 14 , wherein said MLL partner genes are selected from the group consisting of LAF-4, AF4 (MLLT2, FEL), AF5α, AF5q31, AF6q21 (FKHRL1), AF9 (MLLT3), AF10, MLL, AF17, ENL (MLLT1, LTG19), AFX, CBP, ELL (MEN), p300, AF3p21, LCX (TET1), AF15q14, AF1p (eps1S), AF1q, GMPS, LPP, GRAF, AF6, CDK6, FBP17, ABI-1, CBL, MPFYVE, GAS7, LASP1, MSF, EEN, hCDCrel, SEPTIN6, CALM, LARG, GPHN, MYO1F, Alkaline Ceramidase, RPS3, and MIFL.  
     
     
         16 . The method of  claim 5 , wherein said microarray comprises oligonucleotide sequences from MLL partner genes.  
     
     
         17 . The method of  claim 16 , wherein said MLL partner genes are selected from the group consisting of LAF-4, AF4 (MLLT2, FEL), AF5α, AF5q31, AF6q21 (FKHRL1), AF9 (MLLT3), AF10, MLL, AF17, ENL (MLLT1, LTG19), AFX, CBP, ELL (MEN), p300, AF3p21, LCX (TET1), AF15q14, AF1p (eps15), AF1q, GMPS, LPP, GRAF, AF6, CDK6, FBP17, ABI-1, CBL, MPFYVE, GAS7, LASP1, MSF, EEN, hCDCrel, SEPTIN6, CALM, LARG, GPHN, MYO1F, Alkaline Ceramidase, RPS3, and MIFL.  
     
     
         18 . The method of  claim 9 , wherein the microarray comprises oligonucleotide sequences comprising SEQ ID NOs 1-162.  
     
     
         19 . The method of  claim 9 , wherein the microarray comprises oligonucleotide sequences comprising SEQ ID NOs 163-246.  
     
     
         20 . The method of  claim 1 , wherein said isolating of DNA topoisomerase II-DNA complexes of step b) comprises lysing said cells and immunoprecipitating said DNA topoisomerase 11-DNA complexes.  
     
     
         21 . A microarray comprising MLL bcr oligonucleotide sequences.  
     
     
         22 . The microarray of  claim 21 , wherein said MLL bcr oligonucleotide sequences hybridize to non-repetitive MLL bcr sequences.  
     
     
         23 . The microarray of  claim 21 , wherein said microarray further comprises oligonucleotide sequences from the Alu region between nucleotide positions 663-1779 in the MLL bcr.  
     
     
         24 . The microarray of  claim 21 , wherein said microarray further comprises control sequences which are not involved in MLL translocations.  
     
     
         25 . The microarray of  claim 24 , wherein said control sequences are selected from the group consisting of MLL exon 25, MLL exon 3, GAPDH, c-myc, and bacterial gene sequences.  
     
     
         26 . The microarray of  claim 21 , wherein said microarray further comprises oligonucleotide sequences from MLL partner genes.  
     
     
         27 . The microarray of  claim 26 , wherein said MLL partner genes are selected from the group consisting of LAF-4, AF4 (MLLT2, FEL), AF5α, AF5q31, AF6q21 (FKHRL1), AF9 (MLLT3), AF10, MLL, AF17, ENL (MLLT1, LTG19), AFX, CBP, ELL (MEN), p300, AF3p21, LCX (TET1), AF15q14, AF1p (eps1S), AF1q, GMPS, LPP, GRAF, AF6, CDK6, FBP17, ABI-1, CBL, MPFYVE, GAS7, LASP1, MSF, EEN, hCDCrel, SEPTIN6, CALM, LARG, GPHN, MYO1F, Alkaline Ceramidase, RPS3, and MIFL.  
     
     
         28 . A microarray comprising oligonucleotide sequences from MLL partner genes.  
     
     
         29 . The microarray of  claim 28 , wherein said MLL partner genes are selected from the group consisting of LAF-4, AF4 (MLLT2, FEL), AF5α, AF5q31, AF6q21 (FKHRL1), AF9 (MLLT3), AF10, MLL, AF17, ENL (MLLT1, LTG19), AFX, CBP, ELL (MEN), p300, AF3p21, LCX (TET1), AF15q14, AF1p (eps15), AF1q, GMPS, LPP, GRAF, AF6, CDK6, FBP17, ABI-1, CBL, MPFYVE, GAS7, LASP1, MSF, EEN, hCDCrel, SEPTIN6, CALM, LARG, GPHN, MYO1F, Alkaline Ceramidase, RPS3, and MIFL.  
     
     
         30 . The microarray of  claim 21 , wherein the microarray comprises oligonucleotide sequences comprising SEQ ID NOs 1-162.  
     
     
         31 . The microarray of  claim 21 , wherein the microarray comprises oligonucleotide sequences comprising SEQ ID NOs 163-246.  
     
     
         32 . A kit for performing the method of  claim 1 .  
     
     
         33 . The kit of  claim 32 , comprising: 
 a) an agent and a buffer for lysing cells;    b) a solid support and a buffer for isolating DNA-DNA topoisomerase II complexes;    c) at least one primer and a buffer for PCR amplification of isolated DNA by whole genome amplification;    d) at least one first detectable label for incorporation into products of whole genome amplification;    e) calibrated reference standard DNA comprising a second detectable label;    f) a microarray comprising oligonucleotide sequences from the MLL bcr; and    g) instruction material.