Abstract:
The invention relates to a method of for detecting DNA damage in a tissue sample. The method includes the steps of exposing sample DNA to a tagged DNA-damage binding factor and then shearing the DNA to produce fragments. After separating damaged from undamaged DNA, the two are amplified and differentially labeled. The labeled fragments can be immobilized on a microarray allowing the location and extent of any DNA damage to be determined.

Description:
TECHNICAL FIELD 
     The invention relates to a method for detecting DNA damage in a sample; a diagnostic kit for undertaking the said method, including components thereof; and a screening method for screening compounds to identify either whether they damage DNA or whether damaged DNA is affected thereby. 
     BACKGROUND OF THE INVENTION 
     Maintenance of the structure of the eukaryotic genome involves a series of proteins such as, but not exclusively, enzymes which not only monitor DNA for alterations but also effect repairs to any alterations with a view to maintaining the integrity of the DNA for the purpose of subsequent meiosis or mitosis. 
     Different agents cause different types of DNA damage and, as a result, a different series of proteins have evolved in order to repair the different types of damage. 
     It follows that DNA repair is one of the fundamental processes involved in DNA metabolism and defects in any of the DNA repair mechanisms have major biological consequences, including a significant impact on the well-being of the relevant organism. 
     A main goal since the discovery of the structure of DNA has been to systematically determine the precise molecular mechanisms that mediate DNA function. 
     As improved technologies allow for increasingly high resolution studies of DNA it is becomingly increasingly possible to undertake location analysis of DNA, i.e. analysis that enables the precise location of a given event to be determined within the eukaryotic genome. When one considers that the human cell contains 2 metres of DNA packed within chromatin and that these 2 metres of DNA comprise more than 30,000 genes or 3.2 billion base pairs but one is, nevertheless, able to pinpoint the precise location of a given event (within a 100 base pair degree of accuracy) then one begins to appreciate that location analysis is an important tool in understanding events that take place within the genome. 
     There are a number of agents within the environment which are thought to damage DNA. These agents are both chemical and physical and so comprise genotoxic molecules, typically, man-made, and also physical forces such as electromagnetic radiation such as UV rays and X-rays. 
     UV rays are known to be particularly damaging to humans and are the commonest cause of skin cancer due to epidermal absorption of ultraviolet radiation. For cancer DNA is believed to be the primary target and it has been shown that photochemical reactions involving DNA have been linked to mutagenesis, carcinogenesis and cell death. Damage caused by UV exposure is characterised by the formation of pyrimidine dimers and in particular thymine dimers. Nature has responded by producing an enzyme, photolyase, which reactivates DNA by the direct repair of thymine dimers. It binds to the damaged DNA and by absorbing energy restores the pyrimidine dimers to their former monomeric state. This repair mechanism is found in many forms of eukaryotic life. 
     In an analogous fashion other DNA repair proteins, and in particular enzymes, have evolved to repair other forms of DNA damage. Examples of such enzymes include base excision repair enzymes; enzymes which are responsible for direct reversal of damage, repair of DNA-protein crosslinks, mismatch excision repair, nucleotide excision repair, homologous recombination, non-homologous end joining or modulation of nucleotide pools; DNA polymerases, editing and processing nucleases, also gene products which interfere with the Rad6 pathway or which affect chromatin structure, enzymes or gene products encoded by genes which are defective in diseases associated with sensitivity to DNA damaging agents etc. Table 1 lists examples of genes encoding these enzymes and other proteins  1  along with an indication of their functionality and so the nature of the damage that they repair. Many of these enzymes are highly conserved and so homologues exist in different species. 
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Genes Encoding DNA Repair Enzymes or having a DNA Repair function 
               
             
          
           
               
                 Gene Name 
                   
                 Chromosome 
                 NCBI 
               
               
                 (Synonyms) 
                 Activity 
                 Location 
                 Accession No. 
               
               
                   
               
               
                 Base excision repair 
                 DNA glycosylases: major 
                   
                   
               
               
                   
                 altered base released 
               
               
                 UNG 
                 U 
                 12q24.11 
                 NM 080911 
               
               
                 SMUG1 
                 U 
                 12q13.13 
                 NM 014311 
               
               
                 MBD4 
                 U or T opposite G at CpG 
                 3q21.3 
                 NM 003925 
               
               
                   
                 sequences 
               
               
                 TDG 
                 U, T or ethenoC opposite 
                 12q23.3 
                 NM 003211 
               
               
                   
                 G 
               
               
                 OGG1 
                 8-oxoG opposite C 
                 3p25.3 
                 NM 016821 
               
               
                 MUTYH (MYH) 
                 A opposite 8-oxoG 
                 1p34.1 
                 NM 012222 
               
               
                 NTHL1 (NTH1) 
                 Ring-saturated or 
                 16p13.3 
                 NM 002528 
               
               
                   
                 fragmented pyrimidines 
               
               
                 MPG 
                 3-MeA, ethenoA, 
                 16p13.3 
                 NM 002434 
               
               
                   
                 hypoxanthine 
               
               
                 NEIL1 
                 Removes thymine glycol 
                 15q24.2 
                 NM 024608 
               
               
                 NEIL2 
                 Romoves oxidative 
                 8p23.1 
                 NM 145043 
               
               
                   
                 products of pyrimidines 
               
               
                   
                 Other BER Factors 
               
               
                 APEX1 
                 AP endonuclease 
                 14q11.2 
                 NM 001641 
               
               
                 APEX2 
                 AP endonuclease 
                 Xp11.21 
                 NM 014481 
               
               
                 LIG3 
                 DNA Ligase 
                 17q12 
                 NM 013975 
               
               
                 XRCC1 
                 Ligase accessory factor 
                 19q13.31 
                 NM 006297 
               
               
                 PNKP 
                 Converts some DNA 
                 19q13.33 
                 NM 007254 
               
               
                   
                 breaks to ligatable ends 
               
               
                   
                 Poly (ADP-ribose) 
               
               
                   
                 polymerase (PARP) 
               
               
                   
                 enzymes 
               
               
                 PARP1 (ADPRT) 
                 Protects strand 
                 1q42.12 
                 NM 001618 
               
               
                   
                 interruptions 
               
               
                 PARP2 (ADPRTL2) 
                 PARP-like enzyme 
                 14q11.2 
                 NM 005484 
               
               
                 Direct Reversal of 
               
               
                 Damage 
               
               
                 MGMT 
                 O 6 -meG alkyltransferase 
                 10q26.3 
                 NM 002412 
               
               
                 MGC90512 (ABH2) 
                 1-meA dioxygenase 
                 12q24.11 
                 NM 001655 
               
               
                 DEPC-1 (ABH3) 
                 1-meA dioxygenase 
                 11p11.2 
                 NM 139178 
               
               
                 Repair of DNA- 
               
               
                 protein cross links 
               
               
                 TDP1 
                 Removes covalently 
                 14q32.11 
                 NM 018319 
               
               
                   
                 bound TOP1-DNA 
               
               
                   
                 complexes 
               
               
                 Mismatch excision 
               
               
                 repair (MMR) 
               
               
                 MSH2 
                 Mismatch and loop 
                 2p21 
                 NM 000251 
               
               
                   
                 recognition 
               
               
                 MSH3 
                 Mismatch and loop 
                 5q14.1 
                 NM 002439 
               
               
                   
                 recognition 
               
               
                 MSH6 
                 Mismatch and loop 
                 2p16.3 
                 NM 000179 
               
               
                   
                 recognition 
               
               
                 MSH4 
                 MutS homologues 
                 1p31.1 
                 NM 002440 
               
               
                   
                 specialised for meiosis 
               
               
                 MSH5 
                 MutS homologues 
                 6p21.33 
                 NM 002441 
               
               
                   
                 specialised for meiosis 
               
               
                 PMS1 
                 MutL homologue 
                 2q32.2 
                 NM 000534 
               
               
                 MLH1 
                 MutL homologues 
                 3p22.3 
                 NM 000249 
               
               
                   
                 forming heterodimer 
               
               
                 PMS2 
                 MutL homologues 
                 7p22.1 
                 NM 000535 
               
               
                   
                 forming heterodimer 
               
               
                 MLH3 
                 MutL homologues of 
                 14q24.3 
                 NM 014381 
               
               
                   
                 unknown function 
               
               
                 PMS2L3 
                 MutL homologues of 
                 7q11.23 
                 D38437 
               
               
                   
                 unknown function 
               
               
                 PMS2L4 (PMS6) 
                 MutL homologues of 
                 7q11.21 
                 D38500 
               
               
                   
                 unknown function 
               
               
                 Nucleotide excision 
                 (XP = xeroderma 
               
               
                 repair (NER) 
                 pigmentosum) 
               
               
                 XPC 
                 Binds damaged DNA as 
                 3p25.1 
                 NM 04628 
               
               
                   
                 complex 
               
               
                 RAD23B (HR23B) 
                 Binds damaged DNA as 
                 9q31.2 
                 NM 002874 
               
               
                   
                 complex 
               
               
                 CETN2 
                 Binds damaged DNA as 
                 Xq28 
                 NM 004344 
               
               
                   
                 complex 
               
               
                 RAD23A (HR23A) 
                 Substitutes for HR23B 
                 19p13.13 
                 NM 005053 
               
               
                 XPA 
                 Binds damaged DNA in 
                 9q22.33 
                 NM 000380 
               
               
                   
                 preincision complex 
               
               
                 RPA1 
                 Binds DNA in preincision 
                 17p13.3 
                 NM 002945 
               
               
                   
                 complex 
               
               
                 RPA2 
                 Binds DNA in preincision 
                 1p35.3 
                 NM 002946 
               
               
                   
                 complex 
               
               
                 RPA3 
                 Binds DNA in preincision 
                 7p21.3 
                 NM 002947 
               
               
                   
                 complex 
               
               
                 TF11H 
                 Catalyses unwinding in 
               
               
                   
                 preincision complex 
               
               
                 ERCC3 (XPB) 
                 3′ to 5′ DNA helicase 
                 2q14.3 
                 NM 000122 
               
               
                 ERCC2 (XPD) 
                 5′ to 3′ DNA helicase 
                 19q13.32 
                 NM 000400 
               
               
                 GTF2H1 
                 Core TFIIH subunit p62 
                 11p15.1 
                 NM 005316 
               
               
                 GTF2H2 
                 Core TFIIH subunit p44 
                 5q13.2 
                 NM 001515 
               
               
                 GTF2H3 
                 Core TFIIH subunit p34 
                 12q24.31 
                 NM 001516 
               
               
                 GTF2H4 
                 Core TFIIH subunit p52 
                 6p21.33 
                 NM 001517 
               
               
                 GTF2H5 (TTDA) 
                 Core TFIIH subunit p8 
                 6p25.3 
                 NM 207118 
               
               
                 CDK7 
                 Kinase subunit of TFIIH 
                 5q13.2 
                 NM 001799 
               
               
                 CCNH 
                 Kinase subunit of TFIIH 
                 5q14.3 
                 NM 001239 
               
               
                 MNAT1 
                 Kinase subunit of TFIIH 
                 14q23.1 
                 NM 002431 
               
               
                 ERCC5 (XPG) 
                 3′ incision 
                 13q33.1 
                 NM 000123 
               
               
                 ERCC1 
                 5′incision subunit 
                 19q13.32 
                 NM 001983 
               
               
                 ERCC4 (XPF) 
                 5′incision subunit 
                 16p13.12 
                 NM 005236 
               
               
                 LIG1 
                 DNA joining 
                 19q13.32 
                 NM 000234 
               
               
                 NER-related 
               
               
                 CKN1 (CSA) 
                 Cockayne syndrome; 
                 5q12.1 
                 NM 000082 
               
               
                   
                 needed for transcription- 
               
               
                   
                 coupled NER 
               
               
                 ERCC6 (CSB) 
                 Cockayne syndrome; 
                 10q11.23 
                 NM 000124 
               
               
                   
                 needed for transcription- 
               
               
                   
                 coupled NER 
               
               
                 XAB2 (HCNP) 
                 Cockayne syndrome; 
                 19p13.2 
                 NM 020196 
               
               
                   
                 needed for transcription- 
               
               
                   
                 coupled NER 
               
               
                 DDB1 
                 Complex defective in XP 
                 11q12.2 
                 NM 001923 
               
               
                   
                 group E 
               
               
                 DDB2 
                 Complex defective in XP 
                 11p11.2 
                 NM 000107 
               
               
                   
                 group E 
               
               
                 MMS19L (MMS19) 
                 Transcription and NER 
                 10q24.1 
                 NM 022362 
               
               
                 Homologous 
               
               
                 Recombination 
               
               
                 RAD51 
                 Homologous pairing 
                 15q15.1 
                 NM 002875 
               
               
                 RAD51L1 (RAD51B) 
                 Rad51 homologue 
                 14q24.1 
                 NM 002877 
               
               
                 RAD51C 
                 Rad51 homologue 
                 17q23.2 
                 NM 002876 
               
               
                 RAD51L3 (RAD51D) 
                 Rad51 homologue 
                 17q12 
                 NM 002878 
               
               
                 DMC1 
                 Rad51 homologue, 
                 22q13.1 
                 NM 007068 
               
               
                   
                 meiosis 
               
               
                 XRCC2 
                 DNA break and crosslink 
                 7q36.1 
                 NM 005431 
               
               
                   
                 repair 
               
               
                 XRCC3 
                 DNA break and crosslink 
                 14q32.33 
                 NM 005432 
               
               
                   
                 repair 
               
               
                 RAD52 
                 Accessory factors for 
                 12p13.33 
                 NM 002879 
               
               
                   
                 recombination 
               
               
                 RAD54L 
                 Accessory factors for 
                 1p34.1 
                 NM 003579 
               
               
                   
                 recombination 
               
               
                 RAD54B 
                 Accessory factors for 
                 8q22.1 
                 NM 012415 
               
               
                   
                 recombination 
               
               
                 BRCA1 
                 Accessory factor for 
                 17q21.31 
                 NM 007295 
               
               
                   
                 transcription and 
               
               
                   
                 recombination, E3 
               
               
                   
                 Ubiquitin ligase 
               
               
                 BRCA2 (FANCD1) 
                 Cooperation with RAD51 
                 13q13.1 
                 NM 000059 
               
               
                   
                 essential function 
               
               
                 SHFM1 (DSS1) 
                 BRCA2 associated 
                 7q21.3 
                 NM 006304 
               
               
                 RAD50 
                 ATPase in complex with 
                 5q23.3 
                 NM 005732 
               
               
                   
                 MRE11A, NBS1 
               
               
                 MRE11A 
                 3′exonuclease 
                 11q21 
                 NM 005590 
               
               
                 NBS1 
                 Mutated in Nijmegen 
                 8q21.3 
                 NM 002485 
               
               
                   
                 breakage syndrome 
               
               
                 MUS81 
                 A structure specific DNA 
                 11q13.1 
                 NM 025128 
               
               
                   
                 nuclease 
               
               
                 EME1 (MMS4L) 
                 A structure specific DNA 
                 17q21.33 
                 NM 152463 
               
               
                   
                 nuclease 
               
               
                 EME2 
                 Essenital meiotic 
                 16p13.3 
                 NM 0010865 
               
               
                   
                 endonuclease 1 
               
               
                   
                 homologue 2 
               
               
                 Non-homologous 
               
               
                 end-joining 
               
               
                 G22P1 (Ku70) 
                 DNA end binding 
                 22q13.2 
                 NM 001469 
               
               
                 XRCC5 (Ku80) 
                 DNA end binding 
                 2q35 
                 NM 021141 
               
               
                 PRKDC 
                 DNA-dependent protein 
                 8q11.21 
                 NM 006904 
               
               
                   
                 kinase catalytic subunit 
               
               
                 LIG4 
                 Ligase 
                 13q33.3 
                 NM 002312 
               
               
                 XRCC4 
                 Ligase accessory factor 
                 5q14.2 
                 NM 003401 
               
               
                 DCLRE1C (Artemis) 
                 Nuclease 
                 10p13 
                 NM 022487 
               
               
                 XLF (Cernunnos, 
                 XRCC4-LIG4 interacting 
                 2q35 
                 NM 024782 
               
               
                 NHEJ1) 
                 factor 
               
               
                 Modulation of 
               
               
                 nucleotide pools 
               
               
                 NUDT1 (MTH1) 
                 8-oxoGTPase 
                 7p22.3 
                 NM 002452 
               
               
                 DUT 
                 dUTPase 
                 15q1.1 
                 NM 001948 
               
               
                 RRM2B (p53R2) 
                 P53-inducible 
                 8q22.3 
                 NM 015713 
               
               
                   
                 ribonucleotide reductase 
               
               
                   
                 small subunit 2 
               
               
                   
                 homologue 
               
               
                 DNA polymerases 
               
               
                 (catalytic subunits) 
               
               
                 POLB 
                 BER in nuclear DNA 
                 8p11.21 
                 NM 002690 
               
               
                 POLG 
                 BER in mitochondrial 
                 15q26.1 
                 NM 002693 
               
               
                   
                 DNA 
               
               
                 POLD1 
                 NER and MMR 
                 19q13.33 
                 NM 002691 
               
               
                 POLE 
                 NER and MMR 
                 12q24.33 
                 NM 006231 
               
               
                 PCNA 
                 Sliding clamp for pol 
                 20p12.3 
                 NM 002592 
               
               
                   
                 delta and pol epsilon 
               
               
                 REV3L (POLZ) 
                 DNA pol zeta catalytic 
                 6q231 
                 NM 002912 
               
               
                   
                 subunit, essential 
               
               
                   
                 function 
               
               
                 MAD2L2 (REV7) 
                 DNA pol zeta subunit 
                 1p36.22 
                 NM 006341 
               
               
                 REV1L (REV1) 
                 dCMP transferase 
                 2q11.2 
                 NM 016316 
               
               
                 POLH 
                 XP variant 
                 6p21.1 
                 NM 006502 
               
               
                 POLI (RAD30B) 
                 Lesion bypass 
                 18q21.2 
                 NM 007195 
               
               
                 POLQ 
                 DNA crosslink repair 
                 3q13.33 
                 NM 006596 
               
               
                 POLK (DINB1) 
                 Lesion bypass 
                 5q13.3 
                 NM 016218 
               
               
                 POLL 
                 Gap-filling during non- 
                 10q24.32 
                 NM 013274 
               
               
                   
                 homologous end joining 
               
               
                 POLM 
                 Gap filing during non- 
                 7p13 
                 NM 013284 
               
               
                   
                 homologous end joining 
               
               
                 POLN (POL4P) 
                 DNA crosslink repair? 
                 4p16.3 
                 NM 181808 
               
               
                 Editing and 
               
               
                 Processing 
               
               
                 nucleases 
               
               
                 FEN1 (DNase IV) 
                 5′ nuclease 
                 11q12.2 
                 NM 004111 
               
               
                 TREX1 (DNase III) 
                 3′exonuclease, 3′ 
                 3p21.31 
                 NM 033629 
               
               
                   
                 alternative ORF of the 
               
               
                   
                 TREX1/ATRIP gene 
               
               
                 TREX2 
                 3′ exonuclease 
                 Xq28 
                 NM 007205 
               
               
                 EXO1 (HEX1) 
                 5′ exonuclease 
                 1q43 
                 NM 003686 
               
               
                 SPO11 
                 Endonuclease 
                 20q13.32 
                 NM 012444 
               
               
                 FLJ35220 (ENDOV) 
                 Incision 3′ of 
                 17q25.3 
                 NM 173627 
               
               
                   
                 hypoxanthine and uracil 
               
               
                 Rad6 pathway 
               
               
                 UBE2A (RAD6A) 
                 Ubiquitin-conjugating 
                 Xq24-q25 
                 NM 003336 
               
               
                   
                 enzyme 
               
               
                 UBE2B (RAD6B) 
                 Ubiquitin-conjugating 
                 5q31.1 
                 NM 003337 
               
               
                   
                 enzyme 
               
               
                 RAD18 
                 E3 unbiquitin ligase 
                 3p25.3 
                 NM 020165 
               
               
                 UBE2V2 (MMS2) 
                 Ubiquitin-conjugating 
                 8q11.21 
                 NM 003350 
               
               
                   
                 complex 
               
               
                 UBE2N (UBC13) 
                 Ubiquitin-conjugating 
                 12q22 
                 NM 003348 
               
               
                   
                 complex 
               
               
                 Chromatin Structure 
               
               
                 H2AFX (H2AX) 
                 Histone, phosphorylated 
                 11q23.3 
                 NM 002105 
               
               
                   
                 after DNA damage 
               
               
                 CHAF1A (CAF1) 
                 Chromatin assembly 
                 19p13.3 
                 NM 005483 
               
               
                   
                 factor 
               
               
                 Genes defective in 
               
               
                 diseases associated 
               
               
                 with sensitivity to 
               
               
                 DNA damaging 
               
               
                 agents 
               
               
                 BLM 
                 Bloom syndrome helicase 
                 15q26.1 
                 NM 000057 
               
               
                 WRN 
                 Werner syndrome 
                 8p12 
                 NM 000553 
               
               
                   
                 helicase/3′exonuclease 
               
               
                 RECQL4 
                 Rothmund-Thompson 
                 8q24.3 
                 NM 004260 
               
               
                   
                 syndrome 
               
               
                 ATM 
                 Ataxia telangiectasia 
                 11q22.3 
                 NM 000051 
               
               
                 Fanconi anaemia 
               
               
                 FANCA 
                 Involved in tolerance or 
                 16q24.3 
                 NM 000135 
               
               
                   
                 repair of DNA crosslinks 
               
               
                 FANCB 
                 Involved in tolerance or 
                 Xp22.31 
                 NM 152633 
               
               
                   
                 repair of DNA crosslinks 
               
               
                 FANCC 
                 Involved in tolerance or 
                 9q22.32 
                 NM 000136 
               
               
                   
                 repair of DNA crosslinks 
               
               
                 FANCD2 
                 Involved in tolerance or 
                 3p25.3 
                 NM 033084 
               
               
                   
                 repair of DNA crosslinks 
               
               
                 FANCE 
                 Involved in tolerance or 
                 6p21.31 
                 NM 021922 
               
               
                   
                 repair of DNA crosslinks 
               
               
                 FANCF 
                 Involved in tolerance or 
                 11p14.3 
                 NM 022725 
               
               
                   
                 repair of DNA crosslinks 
               
               
                 FANCG (XRCC9) 
                 Involved in tolerance or 
                 9p13.3 
                 NM 004629 
               
               
                   
                 repair of DNA crosslinks 
               
               
                 FANCL 
                 Involved in tolerance or 
                 2p16.1 
                 NM 018062 
               
               
                   
                 repair of DNA crosslinks 
               
               
                 FANCJ (BRIP1, 
                 BRCA1-associated DNA 
                 17q23.2 
                 NM 032043 
               
               
                 BACH1) 
                 helicase 
               
               
                 FANCM 
                 DNA helicase and 
                 14q21.3 
                 XM 048128 
               
               
                   
                 possible nuclease in the 
               
               
                   
                 XPF-Hef-Mus81 family 
               
               
                 FANCN (PALB2) 
                 PALB2 partner and 
                 16p12.1 
                 NM 024675 
               
               
                   
                 localizer of BRCA2 
               
               
                 FAAP24 (C19orf40) 
                 Fanconi anaemia- 
                 19q13.11 
                 NM 152266 
               
               
                   
                 associated protein, 24 
               
               
                   
                 kDa 
               
               
                 Other identified 
               
               
                 Genes with a 
               
               
                 suspected DNA 
               
               
                 repair function 
               
               
                 DCLRE1A (SNM1) 
                 DNA crosslink repair 
                 10q253. 
                 NM 014881 
               
               
                 DCLRE1B (SNM1B) 
                 Related to SNM1 
                 1p13.2 
                 NM 022836 
               
               
                 RPA4 
                 Similar to RPA2 
                 Xp21.33 
                 NM 103347 
               
               
                 APTX (aprataxin) 
                 Processing of DNA 
                 9p21.1 
                 NM 175073 
               
               
                   
                 single-strand interruption 
               
               
                 NEIL3 
                 Resembles NEIL1 and 
                 4q34.3 
                 NM 018248 
               
               
                   
                 NEIL3 
               
               
                 RECQL (RECQ1) 
                 DNA helicase 
                 12p12.1 
                 NM 002907 
               
               
                 RECDQL5 
                 DNA helicase 
                 17q25.1 
                 NM 00100371 
               
               
                 HEL308 
                 DNA helicase 
                 4q21.23 
                 NM 133636 
               
               
                 RAD52B (RDM1) 
                 Similar to RAD52 
                 17q12 
                 NM 145654 
               
               
                 Other conserved 
               
               
                 DNA damage 
               
               
                 response genes 
               
               
                 ATR 
                 ATM- and I-3K-like 
                 3q23 
                 NM 001184 
               
               
                   
                 essential kinase 
               
               
                 RAD1 
                 PCNA-like DNA damage 
                 5p13.2 
                 NM 002853 
               
               
                   
                 sensors 
               
               
                 RAD9A 
                 PCNA-like DNA damage 
                 11q13.2 
                 NM 004854 
               
               
                   
                 sensors 
               
               
                 HUS1 
                 PCNA-like DNA damage 
                 7p12.3 
                 NM 004507 
               
               
                   
                 sensors 
               
               
                 RAD17 (RAD24) 
                 RFC-like DNA damage 
                 5q13.2 
                 NM 002873 
               
               
                   
                 sensor 
               
               
                 CHEK1 
                 Effector kinase 
                 11q24.2 
                 NM 001274 
               
               
                 CHEK2 
                 Effector kinase 
                 22q12.1 
                 NM 007194 
               
               
                 TP53 
                 Regulation of the cell 
                 17p13.1 
                 NM 000546 
               
               
                   
                 cycle 
               
               
                 ATRIP (TREX1) 
                 ATR-interacting protein 
                 3p21.31 
                 NM 130384 
               
               
                   
                 5′alternative ORF of the 
               
               
                   
                 TREX1/ATRIP gene 
               
               
                 TELO2 (Hclk2, 
                 TEL2, telomere 
                 16p13.3 
                 NM 016111 
               
               
                 kiaa0683) 
                 maintenance 2, homologue 
               
               
                   
               
             
          
         
       
     
     SUMMARY OF THE INVENTION 
     Our investigations have led us to develop a method for location analysis of DNA damage which enables us to determine not only the amount of DNA damage, when DNA is subjected to a particular agent, but also the precise location of the damage and so the functional significance thereof. Our invention, in part, involves the use of microarrays, or chips, which, as the man skilled in the art will appreciate, are solid phase arrays of DNA wherein either the whole genome of a given organism or a selected part thereof, such as a selected gene or genes, is/are deposited in a segmented, but overlapping fashion, on a given substrate. In this way, the whole of the genome, or indeed a specific gene, or part thereof, is represented on the substrate but in a series of manageable, yet overlapping, segments. The hybridisation of sample DNA to the substrate enables the precise location of the sample to be determined having regard to the genome or gene that is deposited on the substrate. Given the resolution of this technology it is possible, dependent upon the species to be examined, to determine the precise location of the sample DNA within 5 (yeast) or 100 (human) base pairs. 
     According to a first aspect of the invention there is therefore provided a method for detecting DNA damage in a tissue sample comprising: 
     1. obtaining a sample of DNA from the tissue sample; 
     2. exposing said DNA to at least one selected DNA damage binding factor, which factor has been tagged with a given binding molecule; 
     3. shearing the DNA to produce fragments thereof; 
     4. precipitating those fragments that include, or are associated with the tagged repair protein by binding the said tag to a selected binding partner in order to isolate fragments of DNA that include, or are associated with, said DNA repair protein; optionally 
     5. amplifying and labelling the precipitated fragments; and 
     6. exposing said optionally amplified and labelled precipitated fragments to a selected microarray under conditions that enable the fragments to hybridise to the array whereby the location of any, or the, DNA damage can be determined having regard to the location of the hybridisation fragments with respect to the array; 
     wherein step (2) may be carried out either before or after step (3). 
     The microarray will be chosen according to the species being examined. Thus, if DNA damage is being detected in yeast, then the array may be a genome wide yeast array whereas if DNA damage in humans is being detected, the array may comprise an array covering the entire human genome or a relevant part of the genome. 
     The DNA-damage binding factor may be any substance, but especially a protein, which binds to DNA at a site of damage. It may be a DNA repair protein, for example a DNA repair enzyme such as photolyase or a protein encoded by one of the DNA repair genes in Table 1. Alternatively, the DNA damage binding factor may be a recognition protein such as an antibody which recognises DNA damage. One example of such a recognition protein is an antibody which binds specifically to cyclobutane pyrimidine dimers (CPDs) or more specifically to thymine dimers and are therefore able to be used for detecting a type of DNA damage induced by UV irradiation. 
     The above methodology enables a person of skill in the art to determine the location of any DNA damage within a sample of DNA, depending upon the number and nature of the DNA damage binding factors that are used in the method. Because the precise location of DNA damage in a genome can be detected, it is then possible, with knowledge of this location, to predict the functional significance of the DNA damage. 
     The tissue sample may be, for example a sample of cancerous tissue being targeted by a DNA damaging drug or normal tissue to be used for comparison with cancerous tissue or to be used in an assay for determining whether an agent causes DNA damage. 
     The DNA may be damaged DNA, for example in the case when the presence, location or amount of a DNA damaging drug is to be detected. Alternatively the DNA may be undamaged, for example when the method is used to determine whether or not an agent causes DNA damage. The term “DNA” is also intended to include DNA packaged as chromatin. In this case, the DNA-damage binding factor must be chosen so that it is able to penetrate the chromatin in order to bind to the damaged sites. 
     The shearing of step (3) above may be carried out using any appropriate shearing techniques such as the use of restriction enzymes or, more preferred, sonication which produces suitably smaller fragments of uniform size of DNA for the purpose of increasing the accuracy of the analysis. 
     In a preferred embodiment of the invention the DNA-damage binding factor is tagged with an immunoreactive binding molecule and part 4 above employs the use of an immunoreactive binding partner. 
     The DNA-damage binding factor may be labelled with at least one molecule that allows the protein, and so the DNA to which it is attached, or with which it is associated, to be determined when said DNA hybridises to the microarray. The label is typically a visual label, for example a chromophore, so that a coloured or light signal is produced when the DNA to which the DNA-damage binding factor is attached, or with which it is associated hybridises to the microarray. 
     When the optional amplification step (5) is employed, labelling may be achieved using primers that incorporate a chromophore label into the amplified material so that an enhanced amount of chromophore labelled DNA is produced. This amplified and labelled DNA is then hybridised to the microarray and the location of the DNA is visualised by observing the whereabouts of the label, for example a fluorescent signal, on the microarray. 
     In the method of the invention said DNA-damage binding factor is typically a DNA recognition protein, for example an antibody, that recognises DNA damage and/or a DNA repair enzyme such as, for example, photolyase or one or more of the enzymes encoded by the genes listed in Table 1 from a preferred species of organism having regard to optimisation of the invention. 
     When the DNA-damage binding protein is a DNA repair enzyme, it may be activated in order to repair any damage to the DNA prior to the optional amplification step (5). Therefore the process further includes the optional step of: 
     4b. allowing the DNA repair protein to repair any of the damaged DNA in the precipitate and/or the supernatant. 
     In many cases, it may also be useful to obtain information about the amount or extent of the DNA damage in a sample. Therefore, the method may further comprise comparing the amount of DNA fragments bound to or associated with the DNA repair protein with the amount of DNA fragments which are not so bound or associated. 
     This may be achieved by repeating step (5) of the method using the supernatant obtained when the DNA fragments are immunoprecipitated in step (4) above. The relative amounts of damaged (precipitate) and undamaged (supernatant) DNA can then be compared. 
     Alternatively, a sample of the sheared fragments obtained from step (3) can be amplified and labelled to give a measure of the total amount of DNA. This total amount can then be compared with the amount of DNA bound to the DNA repair protein. 
     To summarise, the method of the invention may after step (4) further comprise the steps of: 
     4a. taking the supernatant from the precipitation step (4) above; 
     4b. optionally allowing, the DNA repair protein to repair any of the damaged DNA in the precipitate and/or the supernatant; 
     5. amplifying and labelling the precipitated (damaged) fragments with a first label; 
     5a. either before, after or simultaneously with step (5) amplifying and labelling the (undamaged) DNA fragments in the supernatant with a second label which is distinguishable from the first label; and 
     6a. either before, after or simultaneously with step (6) exposing said amplified and labelled supernatant fragments from step (5a) to the microarray under conditions that enable the fragments to hybridise to the array; 
     7. detecting the locations and amounts of the first and second labels on the microarray and determining the location of any, or the, DNA damage and the relative amounts of the damaged and undamaged DNA. 
     As briefly outlined above, in addition to or instead of determining the amount of undamaged DNA in the sample, to determine the total amount of DNA in the sample. 
     Therefore the method may, in addition to or in place of the additional steps outlined above contain the steps of: 
     5b. amplifying and labelling a sample of the fragments obtained from step (3) with a third label; and 
     6b. either before, after or simultaneously with step (6) exposing said amplified and labelled supernatant fragments from step (5b) to the microarray under conditions that enable the fragments to hybridise to the array; 
     7. detecting the locations and amounts of the first, second (if present) and third labels on the microarray and determining the location of any, or the, DNA damage and the relative amounts of the damaged, undamaged and total DNA. 
     The method of the invention may be used for a number of purposes, for example in tracking the use of DNA damaging agents. 
     In some cases, the DNA damaging agent may be a DNA or chromatin binding drug. A number of agents used in the treatment of cancer are DNA binding and damaging agents, for example DNA cutters such as topoiomerase I poisons, for example topotecan, irinotecan and other camptothecin derivatives; topoiomerase II poisons for example etoposide, teniposide, daunomycin, adriamycin, idarubicin and mitoxantrone; bleomycin; DNA binders such as actinomycin D and mithramycin. 
     When the DNA damaging agent is a DNA binding drug, the method of the invention may be used to determine whether the drug has targeted the correct cells and/or the correct region of DNA within the target cells. Therefore the invention additionally comprises a method of determining whether a DNA damaging drug has bound to DNA of target cells, the method comprising carrying out a method as described above, wherein the tissue sample comprises target cells for the DNA binding drug and the presence of the DNA binding drug is indicated by the presence of DNA damage in the target cells. The method may also, of course, be used to determine the presence of drug in non-targeted cells. 
     Preferably, such a method also includes the step of determining whether the DNA damaging drug has bound to and damaged the desired target region of the genomic DNA and in this case, step (6) may further include determining whether the location of any DNA damage is the correct target location for the DNA damaging drug. 
     In addition, for this application, the quantitative methods described above may be used to determine the amount of DNA damaging drug in targeted cells compared with the amount in non-targeted cells. This is particularly useful for drugs which are specifically targeted to certain cell types, for example tumour cells or cells in certain organs where cancer or other diseases are to be treated. For this type of application, the quantitative method of the invention may be carried out twice with the tissue sample for each repetition comprising respectively targeted and non-targeted cells. The amount of DNA damaging drug in the cells will be directly related to the amount of DNA damage detected by the quantitative method of the invention. 
     A further application of the quantitative method is to determine the stability of the drug in the genome and/or its rate of metabolism. In this case, the quantitative method may be carried out at two or more separate times and the amounts of damaged DNA in the sample compared at those times in order to determine the rate of clearance of the drug from the DNA. 
     Our invention therefore enables us to monitor the use of DNA or chromatin binding drugs [eg DNA binding anticancer drugs] to determine whether they are targeting the correct DNA target sequences within the cells. It can also be used quantitatively to determine the extent of DNA damage and therefore the amount of DNA damaging drug in a sample. This enables us to monitor clearance of a drug from a tissue and develop a suitable drug treatment regime which can be individualised for each patient. 
     Therefore, there is also provided a method of determining a drug treatment regime for a patient, the method comprising carrying out a method as just described to determine the stability and/or rate of clearance of a DNA damaging drug in the genome and determining when the level in the DNA sample falls below a required level, at which time it is necessary to administer additional DNA damaging drug to a patient 
     This method may also comprise the step of administering additional DNA damaging drug to the patient when the level in the DNA sample falls below the required level. 
     The method of the present invention may also be used as a screening method to identify compounds which cause DNA damage. In this case, the tissue sample will be chosen from normal cells and the DNA will be undamaged DNA. In this case, the method comprises the initial step of exposing some or all of the DNA to a compound to be tested. Although all of the DNA may be treated with the test compound, it is often advantageous to treat a portion of the DNA and to leave a portion untreated as a control. The DNA damage in the test and control samples can then be compared as a means of eliminating false positive results. When the quantitative methods described above are used it is possible to determine not only whether a test compound causes DNA damage but also the amount of damage which is caused. 
     The methodology also has application for testing compounds to determine whether they have the ability to repair DNA damage. In this case, the DNA repair protein used in the method of the invention will be a test DNA repair protein and the DNA in the sample will be damaged DNA. Advantageously, a quantitative method of the invention will be used. Such a method may comprise the steps of 
     i. a first portion of the sample is treated by a quantitative method of the invention which includes step 4b, i.e. allowing the DNA repair protein to repair any of the damaged DNA in the precipitate and/or the supernatant and the extent of the DNA damage determined; 
     ii. a second portion of the sample is treated by a quantitative method of the invention which does not include step 4b and the extent of DNA damage determined; and 
     iii. the extent of the damage in the two samples is compared to give a measure of the ability of the test DNA repair protein to repair damaged DNA. 
     Alternatively or additionally, the results can be compared with the results obtained when a reference DNA repair compound is used. 
     The invention also relates to a kit for carrying out the method and therefore in a further aspect of the invention there is provided a diagnostic kit for detecting DNA damage in a biological sample wherein the kit comprises at least one DNA damage-binding protein that is tagged with a binding partner; 
     a precipitating agent which is adapted to bind with said tagged protein; and 
     a signalling agent or system which enables the DNA damage-binding protein to be visualised when any of the damaged DNA is hybridised to a microarray. 
     Other preferred features are as described above in relation to the method. 
     The kit may also provide a microarray to which the labelled DNA can be hybridised. 
     Our invention therefore enables us to screen for agents that damage DNA or agents that affect damaged DNA such as therapeutics; measure both the extent and the location of DNA damage; and measure the kinetics of DNA repair and or drug stability in the genome. These factors will enable us to identify within a given population, or even within a given individual, agents that are likely to affect the genome and so the health of a population, or individual; genes that are of particular importance for a given pathway and so, when damaged, lead to the breakdown of the pathway and the consequent diseases, for example, the invention could be used to identify a specific oncogene or to correlate a specific oncogene with a specific tumour type which in turn, has diagnostic implications for the population or individual concerned. 
     Our screening method also enables us to screen for potentially carcinogenic agents or indeed any pharmaceutical or chemical agents which need to be tested prior to their release and use. For example, we envisage that our invention may be suitably used when assaying for the potential of any new pharmaceutical product prior to its authorisation for use. Exposure of the human genome to the potential pharmaceutical and an assay for detecting any subsequent DNA damage would be a beneficial regulatory step because it would show whether the pharmaceutical in question had the ability to damage DNA and, if it did, the nature, extent and the reversibility of the DNA that was damaged. 
     We therefore believe that our invention is useful not only as a research tool but as a screening method for screening new and existing agents and a regulatory method for determining the safety of pharmaceuticals and chemicals prior to their subsequent use. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       An embodiment of the invention will now be described by way of example only with reference to the following Figures wherein: 
         FIG. 1 . illustrates the basic protocol for detection of pyrimidine dimers (CPDs) in DNA or chromatin. Cells are irradiated with UV to induce DNA damage (CPDs), following which, chromatin is isolated and sheared by sonication. UV-induced CPDs are detected in the DNA or chromatin using a tagged CPD specific antibody and then the labelled fragments containing CPDs are separated by immunoprecipitation (IP) from damage free DNA (supernatant). DNA is purified from IP and supernatant and amplified and labelled according to the Agilent protocol. The labelled IP samples can be hybridized to microarrays using a combination of supernatant or input samples. Finally the image is obtained by microarray scanner. 
         FIG. 2 : is part of an image generated from the data provided by a microarray scanner, following scanning of a Chip on Chip experimental array. It shows data for the distribution of UV induced CPDs in yeast in a small section of the yeast genome (line entitled “CPD distribution”); data showing the changes in CPD levels in yeast DNA following three hours of repair after UV exposure in the same small section of the genome (line entitled “CPD repair”); and data showing the changes in histone H3 acetylation in response to UV for the same small section of the yeast genome (line entitled “H3Ac level change”). 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     In the following example, the inventors have exemplified the method of the invention using an antibody which binds to CPDs. However, it should be stressed that the method would work equally well using any other DNA-damage binding factor. Examples of such proteins are well known and genes encoding these proteins are listed in Table 1. In addition, the inventors have also conducted experiments using photolyase from yeast as the DNA damage binding factor. 
     General Protocol 
     
         
         
           
             The basic protocol for detection of CPD in chromatin context is outlined in the  FIG. 1 . In addition to detecting CPDs on chromatin, this method also allows one to identify other repair proteins bond to chromatin with specific antibodies. The key stages are: 
           
         
       
    
     1 UV damage cells 
     2 Tagged protein binds DNA damage 
     3 Extract chromatin 
     4 Shear chromatin 
     5 Isolate damaged DNA from undamaged DNA using anti-Tag antibody. 
     6 DNA purification 
     7 Amplification 
     8 labelling 
     From this point it is possible to continue the procedure in a number of ways. The diagram illustrates the various ways that the protocol can be developed. The standard Agilent procedure involves comparing signal from the immunoprecipitated material with the total Input material. Our preferred method is to compare the ratio of the immunoprecipitated chromatin with that which remains in the supernatant (see  FIG. 1 ). This enables us to accurately measure the extent of DNA damage following exposure to UV radiation. This ratio will change as the removal of DNA damage occurs during DNA repair. It is also possible then to compare the material in the supernatant following immunoprecipitation with the true input (i.e sheared and tagged DNA before immunoprecipitation) as an additional control.
         Basic protocol for direct detection of CPD in DNA is similar and can be summarised as follows: the key stages are:       

     1 UV damage cells 
     2 DNA extraction 
     3 Shear DNA by sonication 
     4 Separate damaged DNA (IP) from undamaged DNA (Supernatant) using anti-CPD antibody. 
     5 Amplification and labelling 
     Example 1 
     Chromatin Immunoprecipitation (ChIP) on Chip Protocol and Visualisation of CPD Distribution, CPD Repair after 3 Hours of Treatment with Photolyase and Histone A3 Acetylation Changes 
     DNA Damage 
     Cells were exposed to UV radiation to induce DNA damage in a form including cyclobutane pyrimidine dimer (CPD) formation. 
     Preparation of Chromatin 
     Following the UV treatment, DNA was extracted either as naked DNA or as chromatin according to known methods. The extracted DNA or chromatin was then sheared by sonication. The protocol used for chromatin extraction and sonication is set out below. 
     1. Grow 100 ml cells to 2×10 7  cells/ml over night. 
     2. Add 3 ml of 37% formaldehyde to 100 ml culture. Shake for 10 min at r.t. 
     3. Stop cross-linking by adding 5.5 ml of 2.5M glycine. Shake for 5 min. 
     4. Collect cells, wash once with ice-cold PBS, and once with cold FA/SDS (+PMSF) buffer. Transfer 
     the cells into 2 ml eppendorf during the wash. 
     5. Resuspend cells in 500 μl of FA/SDS (+PMSF) buffer. 
     6. Add 0.5 ml glass beads. Vortex with 2 ml turbomixer at 4° C. for 10 min. 
     7. Puncture a hole at the bottom of the eppendorf with red-hot needle, sit the 2 ml tube on the top 
     of the 15 ml Corning tube. Spin at 2000 rpm for 2 min. Wash the beads 2×250 μl of FA/SDS 
     (+PMSF) buffer. 
     8. Transfer the lysate into 2 ml eppendorf. Spin with a Beckman bench top centrifuge at 4° C. for 
     20 min. Remove the supernatant by aspiration. 
     9. Resuspend the pellets with ˜900 μl of FA/SDS (+PMSF) buffer. Transfer 1000 μl of the 
     suspension into 15 ml Corning tube. 
     10. Sonicate the cell lysate with Biorupter: power “H”, 30 s on 30 s off at 4° C. for 6 cycles. 
     11. Spin at maximum speed for 10 min on a bench top centrifuge. Transfer the supernatant into 
     1.5 ml eppendorf and spin again at 4° C. for 20 min. Collect the supernatant (WCE). Flash freeze 
     with liquid N 2 . 
     Preparation of the Dynabeads (Mouse IgG) 
     The next step of the process was the detection of the CPDs induced in the DNA by the UV irradiation. This was achieved by reacting the fragments with a CPD-specific antibody. A number of CPD specific antibodies are known but the one used in this experiment was the anti-thymine dimer clone KTM53, available from Kamiya Biomedical Company, Seattle, Wash., USA. The process used for labelling the damaged DNA or chromatin fragments is set out below.
     1. Take 50 μl of Dynabeads per sample.   2. 3 times washes with 500 μl PBS BSA0.1% (4° C.)/sample. Mix with vortex and quick spin.   3. Resuspend the beads in 100 μl PBS BSA0.1% per sample.   4. Add 2.5 μl of antibody (depend on individuals).   5. Incubate at 30° C. for 30 min at 1300 rpm in a Eppendorf Thermomixer.   6. Wash 3 times with 500 μl of PBS BSA0.1% (4° C.)/sample.   7. Resuspend the beads in 50 μl of PBS BSA0.1%. Separate into individual tubes depend on the sample numbers. Take the supernatant out.
 
Immunoprecipitation
   

     The next step in the process is the immunoprecipitation of the labelled fragments containing the damaged DNA (CPDs) and the separation of damaged from undamaged DNA fragments. This was achieved by immunoprecipitation using the following protocol. 
     1. Defrost the chromatin sample on ice. Transfer 100 μl of the samples into the washed beads as IP sample, 20 μl into new tube for input. 
     2. Add 50 μl of 10×PBS BSA (10 mg/ml) into the samples, add PBS to 500 μl. Incubate for 2 h at 1300 rpm at 21° C. in a Eppendorf Thermomixer. 
     3. Wash with 500 μl of FA/SDS. 
     4. Wash 3 times with 1 ml of FA/SDS+NaCl (adjust the NaCl concentration to 500 mM). 
     5. The final wash is in an Eppendorf Thermomixer 10 min, 1300 rpm, 21° C. 
     6. Wash with 500 μl of Li solution. 
     7. Wash with 500 μl of cold TE. Pipetting out the solution. 
     8. Elute DNA with 125 μl of Pronase buffer at 65° C. at 900 rpm for 20 min. 
     9. Transfer the supernatant into a new tube. Add 6.25 μl of Pronase (20 mg/ml, H 2 O). incubate at 37° C. for 1 h, then at 65° C. in water bath O.N. 
     10. To the Input (IN) samples (20 μl), add 80 μl of TE, 25 μl of 5× Pronase buffer, 6.25 μl of Pronase, then same as step 9. 
     11. Add 1 μl of 10 mg/ml of RNase to IP and IN samples. Incubate at 37° C. for 1 h. 
     12. Purifying the samples using Qiagen PCR purification kit. 50 μl (30+20) elution, collect all elute. 
     13. Take 5 μl samples for RT-PCR check. 
     Following the immunoprecipitation, both the damaged (precipitated) and the undamaged (supernatant) DNA were amplified using ligase-mediated PCR (LMPCR) according to the following method. 
     Blunt End 
     1. 40 μl of IP sample, 40 μl of IN sample (800× dilution with water). Keep the samples on ice. 
     
       
         
               
               
               
             
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 1× 
                 5× 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 T4 DNA Pol 
                  11 μl 
                  55 μl 
               
               
                   
                 Buffer 
               
               
                   
                 BSA (10 mg/ml) 
                 0.5 μl 
                 2.5 μl 
               
               
                   
                 dNTP (10 mM) 
                 1.0 μl 
                 5.0 μl 
               
               
                   
                 T4 DNA 
                 0.2 μl 
                 1.0 μl 
               
               
                   
                 polymerase 
               
               
                   
                 H 2 O 
                 57.8 μl  
                 289 μl  
               
               
                   
                 Total 
                  70 μl 
                 350 μl  
               
               
                   
                   
               
             
          
         
       
     
     Add 70 μl of the mix. Mix with pipetting, incubate at 12° C. (water bath) for 20 min. 
     2. Add 11.5 μl NaAc (3M pH5.2), 0.5 μl Glycogen (20 mg/ml). Mix with pipette. 
     3. Add 120 μl phenol/chloroform, vortex. Centrifuge 5 min at 15000 rpm at r.t. 
     4. Transfer the upper phase into a new tube. 
     5. Precipitate with 230 μl EtOH (−20° C.). Centrifuge at 15000 rpm for 15 min at 4° C. 
     6. Wash the pellet with 500 μl of cold 75% EtOH. Centrifuge for 5 min. Using pipette to remove the supernatant, avoiding the loss of the pellet. 
     7. Using Speed-Vac to dry the pellet for 7˜min. 
     Ligation 
     Resuspend the pellets in 25 μl of H 2 O. Add 25 μl Mix for ligation. 
     
       
         
               
               
               
             
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 1× 
                 5× 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 H 2 O 
                  13 μl 
                 65 μl 
               
               
                   
                 DNA ligase 
                   5 μl 
                 25 μl 
               
               
                   
                 buffer 
               
               
                   
                 linker hybride 
                 6.7 μl 
                 33.5 μl   
               
               
                   
                 T4 DNA ligase 
                 0.5 μl 
                 2.5 μl  
               
               
                   
                 Total 
                  25 μl 
                 125 μl  
               
               
                   
                   
               
             
          
         
       
     
     Mix with pipetting, incubate at 16° C. (water bath) for O.N. 
     LM-PCR 
     1. Add 6 μl of NaAc ( 3 M) to the ligation mixture. 
     2. precipitation with 130 μl EtOH (100%, ˜20° C.). 
     3. Centrifugation 15 min, 15000 rpm, 4° C. 
     4. Wash with 500 μl EtOH (75%, cold). Spin for 5 min. 
     5. Dry by Speed-Vac for 7 min. 
     6. Resuspend the pellets in 25 ml of H 2 O. 
     7. Add 15 μl PCR mix A 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Mix A (first PCR) 
                 1× 
                 5× 
               
               
                   
                   
               
             
             
               
                   
                 5× HFBuffer(Phusion) 
                   8 μl 
                   40 μl 
               
               
                   
                 dNTP (10 mM) 
                 1.25 μl 
                 6.25 μl 
               
               
                   
                 Oligo 102 (40 μM) 
                 1.25 μl 
                 6.25 μl 
               
               
                   
                 dd H 2 O 
                  4.5 μl 
                 22.5 μl 
               
               
                   
                 Total 
                   15 μl 
                   75 μl 
               
               
                   
                   
               
             
          
         
       
     
     
       
         
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Mix B (first PCR) 
                 1× 
                 5× 
               
               
                   
                   
               
             
             
               
                   
                 dd H 2 O 
                 7 μl 
                 35 μl 
               
               
                   
                 5× HFBuffer(Phusion) 
                 2 μl 
                 10 μl 
               
               
                   
                 Phusion Pol 
                 1 μl 
                  5 μl 
               
               
                   
                 Total 
                 10 μl  
                 50 μl 
               
               
                   
                   
               
             
          
         
       
     
     PCR program PH-LM-15 (For Phusion DNA Pol, NEB) 
                                                                                                                       Step       Time   Temp                                        1   4   min   55° C.           2   3   min   72° C.           3   1   min   98° C.           4   10   s   98° C.           5   30   s   55° C.           6   1   min   72° C.                7       go to 4, 14×                    8   5   min   72° C.                        
8. Pause the Step 1 after 2 min running, add 10 μl of Polymerase Mix B, mix with pipetting, resume the cycles.
 
9. Add 475 μL ddH20 (total volume approximately 525 μL).
 
10. Put 5 μL of the resulting PCR product into a PCR tube (0.2 to 0.5 mL) for a second expansion.
 
                                                 Mix for 2nd PCR   1×   5×                           5× HFBuffer(Phusion)   10 μl    50 μl           dNTP (10 mM)   1.25 μl     6.25 μl            Oligo 102 (40 μM)   1.25 μl     6.25 μl            Phusion DNA Pol   0.5 μl      2.4 μl           dd H 2 O   32 μl   160 μl           Total   45 μl   225 μl                        
11. Put 45 μL of PCR mix to individual PCR tubes.
 
12. Run the PCR program PH-LM-25 below in a thermocycler:
 
                                                                                                                       Step       Time   Temp                                        1   1   min   98° C.           2   10   s   98° C.           3   30   s   55° C.           4   1   min   72° C.                5       go to 2, 24×                    6   5   min   72° C.                        
13. Add 250 μL precipitation mix to each tube.
 
                                                 Precipitation Mix   1×   5×                           Ammonium Acetate (7.5M)    25 μl    125 μl           Ethanol   225 μl   1125 μl           Total   250 μl   1250 μl                        
14. Cool for 30 minutes at −80° C.
 
15. Spin at 20,000×g for 10 minutes at 4° C. to pellet DNA.
 
16. Wash the pellets with 500 μL of 70% EtOH.
 
17. Dry the pellets for 10 minutes with a vacuum dessicator, and resuspend each pellet in 50 μL H2O.
 
18. Measure DNA concentration with NanoDrop (NanoDrop Technologies) (use 10-fold dilutions, if necessary) and normalize all samples to 100 ng/μL.
 
Labelling
 
     Next, the damaged and undamaged DNA samples were differentially labelled (in the dark) using the Invitrogen BioPrime® Total Genomic Labeling System according to the following protocol. 
     1. Genomic DNA samples should be prepared in TE or in water with EDTA in a volume of 22 μl. 
     
         
         
           
             DNA sample in water 17.6 μl 
             5 mM EDTA 4.4 μl 
             Total 22 μl
 
2. Add Alexa Fluor 3 2× Reaction Mix 25 μl; or
 
             Alexa Fluor 5 2× Reaction Mix 25 μl into 22 μl DNA samples (total 47 μl).
 
3. Gently pipette up and down to mix and incubate at 95° C. in dark for 5 min. Immediately cool on ice for 5 min.
 
4. On ice, add 3 μl of Exo-Klenow Fragment to each tube (total 50 μl).
 
5. Vortex tubes briefly and centrifuge to collect the contents.
 
6. Incubate at 37° C. for 2 hours in a heat block in dark.
 
7. After the incubation, if you are storing the reaction for any length of time prior to purification, add 5 μl of 0.5M EDTA to each tube to quench the reaction. Or you can proceed directly to purification step using a invitrogen column.
 
8. Purification with invitrogen column following the instruction. Elute the DNA in 55 μl Blution Buffer E1.
 
9. Take 5 μl of the sample for O.D. check at 260, 280, 320, 555, 650, 750 nm.
 
10. Combine the cy5 and cy3 pair together to 100 μl volume.
 
11. To the 100 μl of sample, add 12 μl of NaAc ( 3 M), 5 μl of Polyacrylamide (2.5 μg/ml). Add 2.5 volume of EtOH (˜290 μl), mix, 10 min at 80° C.
 
12. Centrifugation 15000 rpm for 15 min. Take the supernatant out by pipetting. The pellets are fragile (blue), be careful.
 
13. Wash with 300 μl EtOH (75%, cold). Spin for 5 min at 15000 rpm.
 
14. Pipetting out the supernatant, 5 min Speed-Vac drying. Store at −20° C.
 
Hybridization to Agilent® Chip
 
           
         
       
    
     Finally, the labelled samples were hybridized to an Agilent® chip Containing yeast genome wide arrays. This was achieved using the following method. 
     1. prepare the hybridization mix; 
                                                                                                       Stock   Final concent.   Vol. for 1×                                Na-MES pH 6.9 (500 mM)   50   mM     50 μl           NaCl (5M)   500   mM     50 μl       EDTA (0.5M)   6   mM     6 μl            Sarcosine (20%)   0.5%   12.5 μl           Formamide   30%    150 μl            Herring Sperm DNA   250   ng     1 μl   (BD)       (250 μg/μl)       Yeast tRNA (4.3 μg/μl)   80   μg   18.6 μl   Invitrogen                       (Gibco)       H 2 O           207.9 μl                     
2. Resuspend the pellets with 4 μl H2O. Add 496 μl of the Mix.
 
3. Heat at 95° C. for 3 min.
 
4. incubate at 40° C. for 15 min in the oven.
 
5. Centrifuge briefly.
 
6. Mount the hybridization chamber.
 
7. Pipetting out 500 μl sample on the cover slide. Carefully lay the chip down to touch the liquid and make sure no bubbles form (Agilent side face down).
 
8. Close the chamber. Incubate in the oven for 17±2 hours at 40° C.
 
Wash the Chip
 
Array washing buffer I
         SSPE 20×300 ml   Sarcosine 20% 250 μl   H 2 O to 1 L (700 ml)
 
Array washing buffer II
   SSPE 20×3 ml   H 2 O to 1 L (997 ml)
 
1. Take the hybridization chamber out the oven, transfer the slides to a container filled with washing buffer I. Separate the slides inside the washing solution with a pair of tweezers.
 
2. Rinse the chip slide in the washing solution I for seconds and transfer carefully to a slide rack.
 
3. Soak the rack in a washing buffer I container. Covered in foil, leave it on shaking platform for 5 min with gently shaking (60 rpm).
 
4. Transfer the rack from buffer I to a container with buffer II. Covered in foil, leave it on shaking platform for 5 min with gently shaking (60 rpm).
 
5. Take the rack out from the buffer II very slowly, keep no drop of liquid on the surface of the slide. Use tissue paper to dry the edge of the slide.
       

     Similar experiments have also been conducted using photolyase derived from yeast as the DNA damage binding factor. In these experiments, the bound photolyase was activated in one sample of the immunoprecipitated damaged DNA and the repair process allowed to proceed for three hours. The remainder of the procedure was then carried out as described above for both the unrepaired sample and the repaired sample as well as the undamaged (supernatant) DNA. 
     We have generated genome wide data for the distribution of UV induced CPDs in yeast and this is shown for a small section of the yeast genome in  FIG. 2  (line entitled “CPD distribution”). Present technology theoretically allows the location of CPDs to be detected to an accuracy of 5 base pairs. We have also measured the changes in CPD levels in yeast DNA following three hours of repair after UV exposure. Again, we have generated genome-wide data and a exemplary data for a small section of the genome is again shown in  FIG. 2  (“CPD repair”). Comparing the CPD levels at 0 and 3 hours after UV allows an accurate genome-wide view of CPD repair during the period. We also measured the changes in histone H3 acetylation in response to UV. Once again, genome wide data was obtained and  FIG. 2  illustrates the relevant data for a small section of the yeast genome (H3Ac level change). Changes in chromatin or chromatin-associated proteins often, occur in response to DNA damage and during DNA damage repair. 
     The data obtained for yeast demonstrates the principle of the invention but it would also be possible to obtain equivalent data for other species including humans. For example, using the newly available one million feature whole genome human arrays, it would be possible to obtain similar results for the human genome. Because of the increased size of the human genome as compared with the yeast genome, present technology allows the detection of CPDs to within around 100 base pairs in the human genome.