Genomic analysis method
OwnerGreg Firth, Rajesh Muru Odedrainventors
Life of this patent
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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.
Claims21
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.
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.
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.
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.
Dependencies are inferred from the claim text.