Genotyping preferential genetic disorders via characterizing heteroduplex DNA ; allelic variation and disease diagnosis
Inventors
Assignees
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Publication Number
US-7803545-B2
Publication Date
2010-09-28
Expiration Date
Abstract
Heteroduplex band resolution is found to be improved by modifying the induced heteroduplex generator (IHG) molecule such that the identifier is spaced apart from the nucleotide position immediately adjacent to the site of the polymorphism which is to be genotyped. Unexpectedly, it is found that, when such modified IHG molecules are used, the resolution of the induced heteroduplexes is significantly improved.
Core Innovation
The invention relates to induced heteroduplex generator (IHG) molecules engineered for genotyping a target gene sequence. The IHG molecule is a synthetic DNA sequence including at least one nucleotide position corresponding to a known polymorphic site in genomic DNA, and an identifier is provided as a nucleotide insertion relative to the genomic sequence. The identifier is positioned at a distance of at least one base from the nucleotide position corresponding to the known polymorphic site, while the nucleotide(s) between the polymorphic-site position and the identifier are unchanged from the genomic sequence.
The altered spacing of the identifier from the polymorphic site is stated to unexpectedly improve heteroduplex band resolution after heteroduplex formation and resolving the induced heteroduplexes into bands. The selected IHG is selected to provide improved separation of a resolved plurality of bands, enabling easier allele discrimination and potentially shortening electrophoresis times.
The disclosed approach is applied to genotyping polymorphisms, including SNPs, in gene promoters associated with inherited disorders and cancer. The document includes promoter polymorphisms for IL-10 (-1082) and TNF (-238), comparing prior-art IHG0 where the identifier is adjacent to the polymorphic site with IHG1/IHG2/IHG3 where the identifier is moved by 1-3 bases, and indicates improved band separation for the spaced identifier designs.
Claims Coverage
The document contains one independent claim directed to a genotyping method using engineered induced heteroduplex generator (IHG) molecules with an identifier inserted at least one base away from the polymorphic site. The claim’s inventive scope centers on how the identifier is spaced, and how that spacing is used to obtain improved band separation for genotype determination; dependent claims further constrain the polymorphism type, identifier size, and other format/resolution constraints.
Identifier spaced at least one base from the polymorphic site
Providing a population of induced heteroduplex generator (IHG) molecules corresponding to the target gene sequence, where the IHG is a synthetic DNA sequence including at least one nucleotide position corresponding to a known polymorphic site and a nucleotide insertion (“identifier”) relative to the genomic sequence at a nucleotide position spaced by a distance of at least one base from the nucleotide position corresponding to the known polymorphic site, and where the nucleotide(s) between the polymorphic-site nucleotide position and the identifier are unchanged from the genomic sequence.
Identifier insertion defines improved separation by comparison to unspaced IHG
Selecting the IHG with the at least one identifier comprising an insertion of a base to provide improved separation of a resolved plurality of bands by comparing the separation obtained using the IHG molecule with the separation obtained using a corresponding IHG in which the identifier is not so spaced.
Induced heteroduplex formation and band-based genotype analysis
Combining the respective populations of the IHG molecules and the target gene sequence under conditions suitable for heteroduplex formation to obtain induced heteroduplexes, resolving the induced heteroduplexes into bands on a suitable support, and analysing the resolved induced heteroduplexes to determine the genotype of the target gene sequence.
Overall, the claim coverage centers on engineered IHG molecules where the identifier insertion is spaced at least one base from the polymorphic site, and where that spacing is selected to yield improved separation of resolved bands. Induced heteroduplexes are formed between the target gene sequence and the spaced-identifier IHG, then resolved into bands and analysed to determine genotype.
Stated Advantages
Improved heteroduplex band resolution after heteroduplex formation and separation, enabling easier allele discrimination.
Potentially shortened electrophoresis times.
Documented Applications
Genotyping polymorphisms, including SNPs, in genes tied to inherited disorders and cancer, including IL-10 (-1082) promoter polymorphism and TNF (-238) promoter polymorphism.
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