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Targeted isolation of cloned genomic regions by recombineering for haplotype phasing and isogenic targeting

Technische Universitaet Dresden, Genomics, BioInnovationsZentrum, Dresden, Germany.
Nucleic Acids Research (Impact Factor: 9.11). 08/2011; 39(20):e137. DOI: 10.1093/nar/gkr668
Source: PubMed

ABSTRACT Studying genetic variations in the human genome is important for understanding phenotypes and complex traits, including rare personal variations and their associations with disease. The interpretation of polymorphisms requires reliable methods to isolate natural genetic variations, including combinations of variations, in a format suitable for downstream analysis. Here, we describe a strategy for targeted isolation of large regions (∼35 kb) from human genomes that is also applicable to any genome of interest. The method relies on recombineering to fish out target fosmid clones from pools and thereby circumvents the laborious need to plate and screen thousands of individual clones. To optimize the method, a new highly recombineering-efficient bacterial host, including inducible TrfA for fosmid copy number amplification, was developed. Various regions were isolated from human embryonic stem cell lines and a personal genome, including highly repetitive and duplicated ones. The maternal and paternal alleles at the MECP2/IRAK 1 loci were distinguished based on identification of novel allele-specific single-nucleotide polymorphisms in regulatory regions. Additionally, we applied further recombineering to construct isogenic targeting vectors for patient-specific applications. These methods will facilitate work to understand the linkage between personal variations and disease propensity, as well as possibilities for personal genome surgery.

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    • "For example, bacteriophage lambda-red based homologous recombination in bacteria has simplified genomic DNA modification [19], but has the prerequisite of a library of mapped BACs or plasmid vectors as a source of genomic DNA. The library requirement hinders its application in, for instance, targeting campaigns of unique human iPS cell lines or in patient-specific gene-therapy, although substantial improvements have been made here as well [20]. Moreover, the lambda-red methodology requires sequential steps in targeting vector construction. "
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    ABSTRACT: Efficient gene targeting in embryonic stem cells requires that modifying DNA sequences are identical to those in the targeted chromosomal locus. Yet, there is a paucity of isogenic genomic clones for human cell lines and PCR amplification cannot be used in many mutation-sensitive applications. Here, we describe a novel method for the direct cloning of genomic DNA into a targeting vector, pRTVIR, using oligonucleotide-directed homologous recombination in yeast. We demonstrate the applicability of the method by constructing functional targeting vectors for mammalian genes Uhrf1 and Gfap. Whereas the isogenic targeting of the gene Uhrf1 showed a substantial increase in targeting efficiency compared to non-isogenic DNA in mouse E14 cells, E14-derived DNA performed better than the isogenic DNA in JM8 cells for both Uhrf1 and Gfap. Analysis of 70 C57BL/6-derived targeting vectors electroporated in JM8 and E14 cell lines in parallel showed a clear dependence on isogenicity for targeting, but for three genes isogenic DNA was found to be inhibitory. In summary, this study provides a straightforward methodological approach for the direct generation of isogenic gene targeting vectors.
    PLoS ONE 09/2013; 8(9):e74207. DOI:10.1371/journal.pone.0074207 · 3.23 Impact Factor
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    • "However, almost all of them can be fully contained in a fosmid-sized construct. Extending the available gDNA libraries and alternative approaches, such as targeted clone retrieval (Nedelkova et al., 2011), can bring us closer to a comprehensive resource. Recently, several new methods for site-specific and targeted homologous recombination have been developed in Caenorhabditis elegans (Robert and Bessereau, 2007; Frøkjaer-Jensen et al., 2008). "
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    • "For many studies, including those at structurally complex regions of the genome, the determination of phase on a more local scale may be more useful and sufficient. These more focussed molecular approaches include SNP-specific extraction of targeted genomic regions [29], recombineering [30], and most frequently, PCR-based techniques such as long PCR, allele-specific PCR and a combination of both [31-33], Linking emulsion PCR [34,35] and Haplotype fusion and ligation haplotyping [36]. PCR-based techniques provide haplotype data on a relatively small scale, with the various methods differing in the way that the phase of variants is resolved. "
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    ABSTRACT: Background Genotyping and massively-parallel sequencing projects result in a vast amount of diploid data that is only rarely resolved into its constituent haplotypes. It is nevertheless this phased information that is transmitted from one generation to the next and is most directly associated with biological function and the genetic causes of biological effects. Despite progress made in genome-wide sequencing and phasing algorithms and methods, problems assembling (and reconstructing linear haplotypes in) regions of repetitive DNA and structural variation remain. These dynamic and structurally complex regions are often poorly understood from a sequence point of view. Regions such as these that are highly similar in their sequence tend to be collapsed onto the genome assembly. This is turn means downstream determination of the true sequence haplotype in these regions poses a particular challenge. For structurally complex regions, a more focussed approach to assembling haplotypes may be required. Results In order to investigate reconstruction of spatial information at structurally complex regions, we have used an emulsion haplotype fusion PCR approach to reproducibly link sequences of up to 1kb in length to allow phasing of multiple variants from neighbouring loci, using allele-specific PCR and sequencing to detect the phase. By using emulsion systems linking flanking regions to amplicons within the CNV, this led to the reconstruction of a 59kb haplotype across the DEFA1A3 CNV in HapMap individuals. Conclusion This study has demonstrated a novel use for emulsion haplotype fusion PCR in addressing the issue of reconstructing structural haplotypes at multiallelic copy variable regions, using the DEFA1A3 locus as an example.
    BMC Genomics 12/2012; 13(1):693. DOI:10.1186/1471-2164-13-693 · 4.04 Impact Factor
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