Genomic analysis method

Abstract

A method of comparing genomic DNA from two individuals who share a phenotype, cutting the DNA into fragments long enough to contain on average one or several polymorphisms; combining the fragments under hybridisation conditions and recovering mismatch-free heterohybrids, wherein adapters resistant to nuclease digestion are ligated to the ends of the genomic fragments. A related method uses pooled genomic DNA from individuals with a common phenotype. Another related method uses restricted nucleic acid fragments likely to contain on average less than one natural polymorphism.

1 . A method of performing genomic analysis by: 
 a) digesting genomic DNA to be compared from two different sources to provide genomic fragments whose average length is greater than the average spacing between natural polymorphisms;    b) combining under hybridisation conditions single strands of the genomic fragments from the two sources;    c) separating heterohybrids from homohybrids; and    d) separating mismatch-free heterohybrids from hybrids with mismatches; 
 which method comprises ligating an adapter to each end of each genomic fragment produced in step a), said adapter being, in double-stranded mismatch-free form, resistant to nuclease digestion.  
   
     
     
         2 . The method of  claim 1  wherein the adapter comprises one or more phosphodiester bonds, selected from phosphorothioate and methylphosphonate, that are nuclease resistant.  
     
     
         3 . The method of  claim 1  or  claim 2 , wherein the adapters of a homohybrid include at least one mismatch, and the adapters of a heterohybrid are perfectly matched.  
     
     
         4 . The method of any one of  claims 1  to  3 , wherein steps c) and d) are performed using a mismatch recognition protein and an exonuclease/endonuclease combination.  
     
     
         5 . A method of performing genomic analysis by: 
 ii) providing genomic DNA, pooled from a plurality of individuals that share a phenotype;    ii) digesting the genomic DNA to provide genomic fragments whose average length is greater than the average spacing between natural polymorphisms;    iii) ligating an adapter to each end of each genomic fragment produced in step ii), said adapter being, when in double-stranded mismatch-free form, resistant to nuclease digestion;    iv) denaturing and re-annealing the mixture of adapter-terminated genomic fragments produced in step iii);    v) removing from the mixture produced in step iv) hybrids containing mismatches and if required amplifying mismatch-free hybrids;    vi) and repeating steps iv) and v) to recover one or a few mismatch-free hybrids associated with the phenotype.    
     
     
         6 . The method of  claim 5 , wherein the one or a few mismatch-free hybrids recovered in step vi) are analysed by hybridisation to reference sequences of nucleic acid.  
     
     
         7 . The method of  claim 5 , wherein a mismatch-free hybrid resulting from step vi) is sequenced.  
     
     
         8 . A method of performing genomic analysis by: 
 i) providing first nucleic acid, pooled from a plurality of individuals that share a phenotype;    ii) digesting the said first nucleic acid to provide fragments whose average length is about equal to or less than the average spacing between natural polymorphisms;    iii) ligating an adapter to each end of each fragment produced in step ii) to form adapter-terminated nucleic acid fragments which are, when in double-stranded mismatch-free form, resistant to nuclease digestion;    iv) denaturing and re-annealing the mixture of adapter-terminated nucleic acid fragments produced in step iii);    v) removing from the mixture produced in step iv) hybrids containing mismatches and if required amplifying mismatch-free hybrids;    vi) repeating steps iv) and v) to recover a first mixture of mismatch-free hybrids;    vii) providing second nucleic acid pooled from a plurality of individuals that do not share the same phenotype;    viii) subjecting the nucleic acid of vii) to the said steps of ii) to vi) to recover a second mixture of mismatch-free hybrids;    ix) combining under hybridisation conditions single strands of the said first mixture of mismatch-free hybrids and the said second mixture of mismatch-free hybrids;    x) and recovering nucleic acid fragments that do not form mismatch-free hybrids and are associated with the phenotype.    
     
     
         9 . The method of  claim 8 , wherein the nucleic acid fragments recovered in step x) are analysed by hybridisation to reference sequences of nucleic acid.  
     
     
         10 . The method of  claim 8 , wherein the nucleic acid fragments receovered in step x) are sequenced.  
     
     
         11 . The method of any one of  claims 8  to  10 , wherein the nucleic acid is genomic DNA.  
     
     
         12 . The method of any one of  claims 8  to  10 , wherein the nucleic acid represents a subset of the genome that is transcribed in a tissue or tissues of interest.  
     
     
         13 . The method of any one of  claims 8  to  12 , wherein step x) is performed by physical separation of mismatched fragments following binding to at least one mismatch specific protein.  
     
     
         14 . The method of any one of  claims 5  to  13 , wherein the adapter comprises phosphodiester bonds, selected from phosphorothioate and methylphosphonate, that are nuclease resistant.  
     
     
         15 . The method of any one of  claims 5  to  14 , wherein in step v) hybrids containing mismatches are removed by means of a mismatch recognition protein and an exonuclease/endonuclease combination.  
     
     
         16 . A set of four oligonucleotides, wherein each oligonucleotide of the set: is complementary to a first other oligonucleotide of the set and forms therewith a hybrid that is resistant to nuclease digestion; and is substantially complementary to a second other oligonucleotide of the set.  
     
     
         17 . The set of  claim 16 , wherein each oligonucleotide comprises one or more phosphodiester bonds selected from phosphorothioate and methylphosphonate.  
     
     
         18 . A kit for performing the method of any one of  claims 1  to  4 , which kit comprises the set of four oligonucleotides together with a ligase and a nuclease.  
     
     
         19 . A kit for performing the method of any one of  claims 5  to  15 , which kit comprises a supply of an adapter which is, when in double stranded form, resistant to nuclease digestion, and a ligase and a nuclease.  
     
     
         20 . The kit as claimed in  claim 19 , wherein the adapter comprises one or more phosphodiester bonds selected from the phosphorothioate and methylphosphonate.  
     
     
         21 . The kit of any one of  claims 18  to  20 , wherein a mismatch recognition protein is also present.