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

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.

A Genome-Scale Resource for In Vivo Tag-Based Protein Function Exploration in C. elegans

“…However, almost all of them can be fully contained in a fosmidsized 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 C. elegans (Robert and Bessereau, 2007; Frøkjær-Jensen et al, 2008).…”

Signatures of Crested Ibis MHC Revealed by Recombination Screening and Short-Reads Assembly Strategy

“…Nedelkova’s vectors were used, and the recombinant procedure was performed as recommended by studies [22] with the following modifications. First, 25 μl aliquots from the PCR positive pools were grown in 1 ml of LB supplemented with tetracycline (tet, 5 μg/ml) and chloramphenicol (cm, 10 μg/ml) overnight at 30°C.…”

“…A recently reported recombination method relies on recombineering to fish out target fosmid clones from pools and thereby circumvents the laborious steps of plating and screening thousands of individual clones with traditional solutions [22]. In this study, based on the genomic fosmid library pools, we adopted recombination screening followed by clone-based sequence and assembly (CSA) to assemble the crested ibis MHC.…”

Direct Cloning of Isogenic Murine DNA in Yeast and Relevance of Isogenicity for Targeting in Embryonic Stem Cells

“…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.…”