Analysis method

Details Analysis method Analysis method

2001-09-24

2003-06-03

Whisenant, Ethan (Department: 1634)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C536S023100, C536S024300

Reexamination Certificate

active

06573053

ABSTRACT:

Linkage mapping of genes involved in disease susceptibility and other traits in humans, animals and plants has in recent years become one of the most important engines of progress in biology and medicine. The development of polymorphic DNA markers as landmarks for linkage mapping has been a major factor in this advance. However, current methods that rely on these markers for linkage mapping in humans are laborious, allowing screening of only at most a few markers at a time. Furthermore, their power is limited by the sparsity of highly-informative markers in many parts of the human genome.
Genomic mismatch scanning (GMS) is a positional cloning strategy that has no requirement for conventional polymorphic markers or gel electrophoresis. It isolates fragments of identity-by-descent (IBD) between two related individuals based on the formation of extensive mismatch-free hybrid molecules. The GMS technique is described in U.S. Pat. No. 5,376,526, and is illustrated in
FIG. 1
of the drawings accompanying this specification.
Dam methylation of one sample prior to hybridisation permits discrimination of homohybrid duplexes by virtue of methylation-sensitive restriction endonucleases that cleave only fully methylated or fully unmethylated DNA. The MutHLS methyl-directed mismatch repair proteins cleave mismatched heteroduplexes on the unmethylated strand. Except for mismatch-free heterohybrid molecules, all DNA is eliminated by a combination of Exonuclease III digestion and physical separation of single-stranded DNA using binding columns. The selected molecules are amplified by inter-Alu PCR using combinations of generic primers, and subsequently identified by hybridisation to an ordered array of DNA samples representing intervals of the genome.
Because natural polymorphisms occur on average once every several hundred bp i.e. at least once every 1000 bp, heterohybrids that are several kilobases in length and mismatch-free are likely to be IBD. Similarly, non-IBD alleles in sufficiently large heteroduplexes are likely to contain one or more mismatches and will be cleaved by the mismatch repair proteins.
The IBD maps from multiple pairs of affected relatives are combined and the resulting composite map searched for loci where genotypic concordance occurs more frequently than would be expected by chance. These loci represent candidate regions that may harbour the target mutation(s).
The relative recovery of DNA from a locus when the two genomes share an allele IBD compared to the recovery from that locus when the two genomes are not IBD dictates the reliability of the technique. For analyses involving human genomic DNA, enrichment by a factor of 1-2 for 50% of IBD fragments and by a factor of 2-5 for 35% of IBD fragments has been reported. Only 15% of IBD fragments are reported to be enriched by a factor of >5. Furthermore, the yield of DNA after GMS selection is very poor such that amplification of the selected fragments prior to hybridisation to the array is required.
Enrichment of Fragments of IBD in Two Individuals with Common Ancestry.
It is one object of the present invention to provide novel methods of performing genetic analysis to obtain enrichment of fragments of IBD. Thus in one aspect this invention provides 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.
The method involves comparing genomic DNA from two different sources, generally two different viral or prokaryote or eukaryote (e.g. human, animal or plant) individuals who share a particular phenotype which may have been acquired from a common ancestor. Phenotypes are observable or measurable characteristics displayed by an organism under a particular set of environmental and/or genetic influences. Hybridisation conditions may depend on the genomic fragments being analysed and will be well known to the skilled reader. As noted, natural polymorphisms occur in human genomic DNA on average once every several hundred bp i.e. at least once every 1000 bp. The genomic DNA of the two individuals to be compared is cut into fragments that are in general longer than this. Thus each genomic fragment contains on average one or more polymorphisms. This may be effected by use of a restriction enzyme (or two or more restriction enzymes) that cuts relatively infrequently. Suitable restriction enzymes include those of type II and also those of type IIS. It would alternatively be possible to effect restriction of the genomic DNA by physical or chemical as opposed to enzymatic means.
An adapter is ligated to each end of each fragment. An adapter is an at least partly double-stranded polynucleotide, generally oligonucleotide, having if required an overhang complementary to the overhang generated by the restriction enzyme. Alternatively, both the fragments and the adapter may have blunt ends for ligation. The adapters may comprise oligonucleotides of an arbitrary sequence that does not render them prone to secondary structure, liable to hinder efficient ligation, amplification, or selection on the basis of mismatch discrimination. Primers, comprising all or part of an adapter sequence, used for amplifying DNA under analysis, are further examined to ensure non-specific amplification is avoided. When in double-stranded mismatch-free form, the adapter is resistant to nuclease digestion, that is to say more resistant than is ordinary DNA. Such resistance can be conferred by providing modified internucleotide linkages e.g. phosphorothioate or methylphosphonate linkages, or by the use of nucleotide analogues that confer nuclease resistance. Preferably however a first adapter ligated to fragments of genomic DNA from the first source contains a mismatch; and a second adapter ligated to fragments of genomic DNA from the second source also contains a mismatch; the two adapters being so designed that the forward strand of one adapter will hybridise to the backward strand of the other adapter to form a mismatch-free heterohybrid. A heterohybrid comprises two strands from different individuals and is contrasted with a homohybrid which comprises two strands from the same individual. The two systems are described in more detail below with reference to
FIGS. 2 and 3
of the accompanying drawings, in which:
FIG. 2
shows the use of two adapters each having a mismatch within a section comprising phosphorothioate linkages; and
FIG. 3
shows the use of two different adapters each having a mismatch outside a section having phosphorothioate linkages.
The modified method for affected-pair analyses involves restriction digestion of both genomic DNA samples and ligation of adapter sequences to each. These adapters contain mismatched regions that persist after hybridisation in the homoduplex molecules. By contrast, the adapter sequences are fully complementary in heteroduplexes. Subsequent use of a mismatch recognition protein e.g. T4 endonuclease VII and nuclease digestion results in the elimination of all molecules possessing mismatches. Mismatch-free heteroduplex molecules are resistant to digestion e.g. due to the inclusion of phosphorothioate or methyphosphonate linkages in the adapter sequences that convey protection. These molecules can be amplified efficiently and conveniently with a single primer pair prior to analysis as discussed below.
The ligation of adapters to all fragment ends provides a convenient opportunity to selectively digest homohybrid molecules that are produced by hybridisation of the two DNA samples, and to amplify efficiently the enriched fragments w

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