Induced heteroduplex generators

#This invention relates to induced heteroduplex generator (IHG) molecules which offer improvements over those known in the art. The invention further relates to methods for the formation of induced heteroduplexes between the improved IHG of the invention and target genomic DNA sequences, and the analysis of such induced heteroduplexes for the genotyping of mutations in animal (e.g. mammalian), plant, viral or bacterial DNA, typically human DNA. The invention may thus be employed, for example, in respect of polymorphisms and mutations within genes associated with inherited genetic disorders (such as for example cystic fibrosis (CFTR gene), phenylketonuria (PAH gene), sickle cell disease and β-thalassaemia (HBB gene)), and also somatic mutations within oncogenes and tumour suppressor genes. The invention also relates to kits comprising the induced heteroduplex generators of the invention, for use in methods of analysing genomic DNA sequences.

#The ability to detect DNA sequence variances (hereinafter referred to as “polymorphisms” for convenience, but without limitation) in an organism's genome has become an important tool in the diagnosis of diseases and disorders and in the prediction of response to therapeutic regimes. It is becoming increasingly possible, using early variance detection, to diagnose and treat, even prevent the occurrence of, a disease or disorder before it has physically manifested itself. Furthermore, variance detection can be a valuable research tool in that it may lead to the discovery of genetic bases for disorders the cause of which were hitherto unknown or thought to be other than genetic.

#Sequence variance can take a number of different forms. For example, a variance can arise through substitution of one or more nucleotides for the same number of others at a particular locus in a gene. Another example would be the deletion of one or more nucleotides from a particular locus in a gene. A still further example would be the insertion of one or more extra nucleotides at a particular locus in a gene. Combinations of substitution, deletion and insertion are also possible. A common type of sequence variance is the single nucleotide polymorphism or SNP. A SNP involves the substitution of one nucleotide for another at a particular locus in a gene. Although each SNP involves only one nucleotide, a single gene may contain numerous SNPs.

#The attached figures are provided purely for illustration and without limiting the scope of the invention.

#FIG. 1 illustrates the heteroduplex patterns obtained using an IHG of the prior art (IHG0) and IHGs of the invention (IHG1, IHG2, and IHG3), in the genotyping of the “−1082” polymorphism in the promoter of the IL-10 gene.

#FIG. 2 illustrates the heteroduplex patterns obtained using an IHG of the prior art (IHG0) and IHGs of the invention (IHG1 and IHG2), in the genotyping of the “−238” polymorphism in the Tumour Necrosis Factor (TNF) gene promoter.

#According to the invention, the synthetic DNA sequence includes at least one nucleotide substitution, deletion and/or insertion relative to the genomic sequence at a nucleotide position spaced by a distance of at least one base from the nucleotide position which corresponds to the known polymorphic site and wherein the nucleotide(s) between the nucleotide position which corresponds to the polymorphic site and the nucleotide substitution, deletion and/or insertion are unchanged from the genomic sequence. Where the genomic sequence includes a plurality of known polymorphic sites, the identifier is spaced by a distance of at least one base from the nucleotide position(s) which correspond to each of the known polymorphic sites.

#The spacing of the nucleotide substitution, deletion and/or insertion from the nucleotide position which corresponds to the known polymorphic site is preferably up to about ten bases, more preferably up to about eight bases, e.g. from 1 to about 6 bases, most particularly any number selected from 1, 2, 3, 4, 5 or 6 bases. In the case where there are two or more known polymorphic site, the preferred spacing of up to 10 bases is in relation to the polymorphic site which is closest to the identifier.

#In accordance with one embodiment of this invention, genetic material of a subject, normally a human subject, is analysed to determine its genotype with respect to a particular single nucleotide polymorphism (SNP) at a specific location of a gene. The gene which includes the SNP has at least two alleles, which are referred to here as the reference allele and the variant allele. The reference allele (prototypical or wild type allele) has been designated arbitrarily and typically corresponds to the nucleotide sequence of the gene which has been deposited with GenBank or TIGR under a given accession number. The variant allele contains a variant nucleotide (that is a different nucleotide to that of the reference allele) at the SNP site or at each of the SNP sites in the gene.