Citation Context

...), which carries in its genome the gbaRecA recombineering operon (32) under the rhamnose inducible promoter (PRHA) (37) as well as the TrfaA protein (38) under the arabinose inducible promoter (PBAD) =-=(39)-=-. The TrfA protein is required for initiation of the replication from the bidirectional origin OriV and subsequent increase in the fosmid copy number. The strain is highly stable (Supplementary Figure...

Citation Context

...rough the insertion of a modified blasticidin cassette flanked by 50-bp long homology arms (Figure 2B). We optimized the method using genomic DNA isolated from H7 human embryonic stem (hES) cell line =-=(40)-=-. Based on the recombineering efficiencies determined with single fosmids (6.8 10 3 recombinants/10 6 cells) and given that the number of surviving cells in a typical recombineering reaction in the ab...

Citation Context

...30). A similar approach to isolate genomic regions in BACs has been published recently (46). We use fosmids, because they are easy to handle, stable, suitable for genomic structural variation studies =-=(2,5,22)-=- and preparation of targeting constructs. Most importantly, compared to BAC libraries, fosmid library construction requires much less genomic DNA, which is a major consideration when the source of DNA...

Citation Context

...time required to generate and map them are often not justified when only a specific region of the genome needs to be investigated. In this study, we present a simple approach, based on recombineering =-=(23,24)-=- for targeted isolation of genomic regions in a vector format, suitable for downstream analysis. Recombineering is a DNA engineering technology, based on homologous recombination in Escherichia coli, ...

Citation Context

...1). Recent advances in high-throughput DNA sequencing technologies have enabled rapid progress in the field (14) and in the near future their detection in personal genomes will be performed routinely =-=(15,16)-=-. However, the variations lying in duplicated and highly identical sequences are still difficult to resolve and extensive bioinformatic analysis is needed to map the short next-generation sequencing r...

Citation Context

...polymorphisms at the maternal and paternal alleles (8). SNPs in intergenic and intronic elements like enhancers have been shown to regulate gene expression (9,10) and to contribute to human disorders =-=(7,11)-=-. Recently, it was demonstrated that the activity of long interspersed elements contributes to inter individual genetic variations and can be associated with disease phenotypes (12,13). Various method...

Citation Context

...30). A similar approach to isolate genomic regions in BACs has been published recently (46). We use fosmids, because they are easy to handle, stable, suitable for genomic structural variation studies =-=(2,5,22)-=- and preparation of targeting constructs. Most importantly, compared to BAC libraries, fosmid library construction requires much less genomic DNA, which is a major consideration when the source of DNA...

Citation Context

...lel sequencing is revealing the diversity of genetic variation within the human genome (1–5). They encompass SNPs, insertions, deletions, inversions and duplications, which can be linked with disease =-=(1,6)-=-. Understanding the genetic architecture of complex traits requires knowledge about the polymorphisms in different parts from the genome, including non-coding regions (6,7) as well as information abou...

Citation Context

...1). Recent advances in high-throughput DNA sequencing technologies have enabled rapid progress in the field (14) and in the near future their detection in personal genomes will be performed routinely =-=(15,16)-=-. However, the variations lying in duplicated and highly identical sequences are still difficult to resolve and extensive bioinformatic analysis is needed to map the short next-generation sequencing r...

Citation Context

...ions lying in duplicated and highly identical sequences are still difficult to resolve and extensive bioinformatic analysis is needed to map the short next-generation sequencing reads in such regions =-=(17,18)-=-. Although the detection of structural variations is very important, base pair resolution of their breakpoints and further functional analysis is usually required to define their potential impact (19,...

Citation Context

...able (Supplementary Figure S1) with rates of spontaneous rearrangements in the absence of induction comparable with the previously published recombineering proficient hosts GB05(BAD)Red (33) or DY380 =-=(34)-=-. We optimized the recombineering conditions using a blasticidin resistance cassette insertion assay into a single fosmid clone (Figure 1A). One of the strands of the dsDNA cassette was phosphorylated...

Citation Context

...Rac prophage (23,25). We and others have shown that recombineering has many applications, including subcloning by gap repair (25), point mutagenesis in BACs (24), oligonucleotide directed mutagenesis =-=(26)-=-, BAC engineering for gene targeting (27,28) or protein tagging (29–31). The high efficiency and fidelity of recombineering permits high-throughput DNA engineering at genome scale (30,31). Here, we de...

Citation Context

... human disorders (7,11). Recently, it was demonstrated that the activity of long interspersed elements contributes to inter individual genetic variations and can be associated with disease phenotypes =-=(12,13)-=-. Various methods exist for genome-wide identification of SNPs and structural variations (1). Recent advances in high-throughput DNA sequencing technologies have enabled rapid progress in the field (1...

Citation Context

...ering is a DNA engineering technology, based on homologous recombination in Escherichia coli, mediated by the phage proteins Reda/Redb or their functional counterparts RecE/RecT from the Rac prophage =-=(23,25)-=-. We and others have shown that recombineering has many applications, including subcloning by gap repair (25), point mutagenesis in BACs (24), oligonucleotide directed mutagenesis (26), BAC engineerin...

Citation Context

...shown that recombineering has many applications, including subcloning by gap repair (25), point mutagenesis in BACs (24), oligonucleotide directed mutagenesis (26), BAC engineering for gene targeting =-=(27,28)-=- or protein tagging (29–31). The high efficiency and fidelity of recombineering permits high-throughput DNA engineering at genome scale (30,31). Here, we demonstrate an application of recombineering f...

Citation Context

...ted mutagenesis (26), BAC engineering for gene targeting (27,28) or protein tagging (29–31). The high efficiency and fidelity of recombineering permits high-throughput DNA engineering at genome scale =-=(30,31)-=-. Here, we demonstrate an application of recombineering for selective isolation of large genomic fragments of choice from complex genomes. It circumvents the need for the classical method of library s...

Citation Context

...lel sequencing is revealing the diversity of genetic variation within the human genome (1–5). They encompass SNPs, insertions, deletions, inversions and duplications, which can be linked with disease =-=(1,6)-=-. Understanding the genetic architecture of complex traits requires knowledge about the polymorphisms in different parts from the genome, including non-coding regions (6,7) as well as information abou...

Citation Context

... can be linked with disease (1,6). Understanding the genetic architecture of complex traits requires knowledge about the polymorphisms in different parts from the genome, including non-coding regions =-=(6,7)-=- as well as information about the haplotype phasing, that is the combination of polymorphisms at the maternal and paternal alleles (8). SNPs in intergenic and intronic elements like enhancers have bee...

Citation Context

... human disorders (7,11). Recently, it was demonstrated that the activity of long interspersed elements contributes to inter individual genetic variations and can be associated with disease phenotypes =-=(12,13)-=-. Various methods exist for genome-wide identification of SNPs and structural variations (1). Recent advances in high-throughput DNA sequencing technologies have enabled rapid progress in the field (1...

Citation Context

...ion in any medium, provided the original work is properly cited.e137 Nucleic Acids Research, 2011, Vol. 39, No. 20 PAGE 2 OF 11 target-enrichment strategies, based on polymerase chain reaction (PCR) =-=(21)-=-, hybridization or molecular inversion probes (15) merely detect variations, without isolation of the intact allele as a clone that can be further analyzed to link polymorphisms over large regions or ...

Citation Context

...plotype phasing, that is the combination of polymorphisms at the maternal and paternal alleles (8). SNPs in intergenic and intronic elements like enhancers have been shown to regulate gene expression =-=(9,10)-=- and to contribute to human disorders (7,11). Recently, it was demonstrated that the activity of long interspersed elements contributes to inter individual genetic variations and can be associated wit...

Citation Context

...3). Various methods exist for genome-wide identification of SNPs and structural variations (1). Recent advances in high-throughput DNA sequencing technologies have enabled rapid progress in the field =-=(14)-=- and in the near future their detection in personal genomes will be performed routinely (15,16). However, the variations lying in duplicated and highly identical sequences are still difficult to resol...

Citation Context

...shown that recombineering has many applications, including subcloning by gap repair (25), point mutagenesis in BACs (24), oligonucleotide directed mutagenesis (26), BAC engineering for gene targeting =-=(27,28)-=- or protein tagging (29–31). The high efficiency and fidelity of recombineering permits high-throughput DNA engineering at genome scale (30,31). Here, we demonstrate an application of recombineering f...

Citation Context

...ted mutagenesis (26), BAC engineering for gene targeting (27,28) or protein tagging (29–31). The high efficiency and fidelity of recombineering permits high-throughput DNA engineering at genome scale =-=(30,31)-=-. Here, we demonstrate an application of recombineering for selective isolation of large genomic fragments of choice from complex genomes. It circumvents the need for the classical method of library s...

Citation Context

...ir polymorphisms at single nucleotide resolution. Exploring the impact of the mutations and their characterization as benign or disease associated can be achieved through gene targeting in stem cells =-=(51,52)-=- with isogenic constructs. Our approach permits generation of such constructs with personal genome specific combination of variations. The isogenicity of the flanking homologous sequences is an import...

Citation Context

...ions lying in duplicated and highly identical sequences are still difficult to resolve and extensive bioinformatic analysis is needed to map the short next-generation sequencing reads in such regions =-=(17,18)-=-. Although the detection of structural variations is very important, base pair resolution of their breakpoints and further functional analysis is usually required to define their potential impact (19,...

Citation Context

...highly repetitive, regions. Indeed, previous work has shown that overexpression of Redg from a plasmid can increase the total number of colonies, but the frequency of correct recombinant BACs was low =-=(48)-=-. Transient RecA co-expression from a plasmid has been previously shown to enhance the total number of colonies surviving electroporation (32), but leaky expression of RecA could cause increased basal...

Citation Context

...d a new fosmid library host (GB05RedTrfA) (Figure 1), which carries in its genome the gbaRecA recombineering operon (32) under the rhamnose inducible promoter (PRHA) (37) as well as the TrfaA protein =-=(38)-=- under the arabinose inducible promoter (PBAD) (39). The TrfA protein is required for initiation of the replication from the bidirectional origin OriV and subsequent increase in the fosmid copy number...

Citation Context

...,18). Although the detection of structural variations is very important, base pair resolution of their breakpoints and further functional analysis is usually required to define their potential impact =-=(19,20)-=-. The existing *To whom correspondence should be addressed. Tel: +49 351 463 40129; Fax: +49 351 463 40143; Email: stewart@biotec.tu-dresden.de Present address: Marcello Maresca, Novartis Institutes f...

Citation Context

...neering as it provides higher efficiency and fidelity (36). Either strand can be used, but the strand annealing to the lagging strand of the replication fork is favored by the recombineering reaction =-=(43)-=-. In our experiments, the efficiencies between the two strands varied several fold (Supplementary Table S6), indicating that testing both strands can be beneficial for the isolation of difficult regio...

Citation Context

...r several E. coli chromosomal locations has previously been correlated with the rate of replication of the regions (26). Previously a method to screen genomic libraries by recombineering was reported =-=(47)-=-. However, this method does not appear to have been subsequently utilized, possibly because the complex counter selection strategy imposed practical difficulties. Similarly our previous experience wit...

Citation Context

... recombined with this cassette) to prevent background recombination. The cassettes were phosphorylated at one 50-end but not to the other 50-end to generate PO or OP cassettes, where O means hydroxyl =-=(36)-=-. The cassettes were purified from the PCR reaction using MSB Spin PCRapace kit (Invitek, Berlin, Germany). The cassette for testing the recombineering efficiency of the E. coli strains was also phosp...

Citation Context

...ively parallel sequencing (1–4). However, knowledge about the ‘haplotype phasing’ in different genomes has been scarce (8). Two recently published methods for genome-wide resolution of the haplotypes =-=(49,50)-=- pave the way to systematically study haplotype phasing in individual genomes and cell lines. Our approach is complementary to these studies and allows for the determination of SNP linkage and therefo...

Citation Context

...n a fosmid clone format suitable for sequencing and genetic engineering. We generated a new fosmid library host (GB05RedTrfA) (Figure 1), which carries in its genome the gbaRecA recombineering operon =-=(32)-=- under the rhamnose inducible promoter (PRHA) (37) as well as the TrfaA protein (38) under the arabinose inducible promoter (PBAD) (39). The TrfA protein is required for initiation of the replication ...

Citation Context

...ir polymorphisms at single nucleotide resolution. Exploring the impact of the mutations and their characterization as benign or disease associated can be achieved through gene targeting in stem cells =-=(51,52)-=- with isogenic constructs. Our approach permits generation of such constructs with personal genome specific combination of variations. The isogenicity of the flanking homologous sequences is an import...

Citation Context

...d genetic engineering. We generated a new fosmid library host (GB05RedTrfA) (Figure 1), which carries in its genome the gbaRecA recombineering operon (32) under the rhamnose inducible promoter (PRHA) =-=(37)-=- as well as the TrfaA protein (38) under the arabinose inducible promoter (PBAD) (39). The TrfA protein is required for initiation of the replication from the bidirectional origin OriV and subsequent ...

Citation Context

...ively parallel sequencing (1–4). However, knowledge about the ‘haplotype phasing’ in different genomes has been scarce (8). Two recently published methods for genome-wide resolution of the haplotypes =-=(49,50)-=- pave the way to systematically study haplotype phasing in individual genomes and cell lines. Our approach is complementary to these studies and allows for the determination of SNP linkage and therefo...

Citation Context

...NANOG genes and their surrounding regions were also successfully captured (Figure 3E and F). NANOG has several pseudogenes and one of them, NANOG P1, arose through local duplication of the NANOG gene =-=(41)-=-. In order to isolate the gene, 100 bp of homology sequence unique to the NANOG locus was chosen. The captured fosmid covers the whole locus, an intergenic region and part of the neighboring gene, whi...

Citation Context

...,18). Although the detection of structural variations is very important, base pair resolution of their breakpoints and further functional analysis is usually required to define their potential impact =-=(19,20)-=-. The existing *To whom correspondence should be addressed. Tel: +49 351 463 40129; Fax: +49 351 463 40143; Email: stewart@biotec.tu-dresden.de Present address: Marcello Maresca, Novartis Institutes f...

Citation Context

...time required to generate and map them are often not justified when only a specific region of the genome needs to be investigated. In this study, we present a simple approach, based on recombineering =-=(23,24)-=- for targeted isolation of genomic regions in a vector format, suitable for downstream analysis. Recombineering is a DNA engineering technology, based on homologous recombination in Escherichia coli, ...

Citation Context

...rge regions or to be genetically manipulated for downstream functional analysis. Allele linkages can be achieved using whole genome bacterial artificial chromosome (BAC) or fosmid DNA clone libraries =-=(12,22)-=- but the costs and time required to generate and map them are often not justified when only a specific region of the genome needs to be investigated. In this study, we present a simple approach, based...

Citation Context

... Restriction analysis confirmed that the highly repetitive fosmids were not rearranged (Supplementary Figure S2 and Supplementary Table S5). In further exercises, we used the male hES cell line Shef4 =-=(42)-=- and a primary leukemic sample. With the available cassettes, we isolated Shef4 MECP2, OCT4, PAX6 and GATA4 regions (Supplementary Table S5). For the leukemic sample, we focused on potential disease-r...

Citation Context

...enotypes, diseases, drug responsiveness and the mechanisms of complex traits (6). For many applications, only a small part of the genome, such as specific genes or regulatory regions, are of interest =-=(44,45)-=-. The current methods for selected enrichment of genomic regions followed by next generation sequencing are based on PCR or hybridization approaches (15). These methods encounter size limitations part...

Citation Context

...plotype phasing, that is the combination of polymorphisms at the maternal and paternal alleles (8). SNPs in intergenic and intronic elements like enhancers have been shown to regulate gene expression =-=(9,10)-=- and to contribute to human disorders (7,11). Recently, it was demonstrated that the activity of long interspersed elements contributes to inter individual genetic variations and can be associated wit...

Citation Context

...asmid and encodes the Redb protein instead of the RedgbaRecA operon. The E. coli strain GB05RedTrfA was constructed by insertion of the double operon PBADTrfA-PRharedgbarecA at the ybcC locus of GB05 =-=(33)-=-. For development of the cassette the PRha promoter was amplified from pRedFlp (30). The PBAD promoter from the PBADredgbarecA operon was replaced with PRha by recombineering. The PBADTrfA was amplifi...

Citation Context

...enotypes, diseases, drug responsiveness and the mechanisms of complex traits (6). For many applications, only a small part of the genome, such as specific genes or regulatory regions, are of interest =-=(44,45)-=-. The current methods for selected enrichment of genomic regions followed by next generation sequencing are based on PCR or hybridization approaches (15). These methods encounter size limitations part...

Citation Context

...n different parts from the genome, including non-coding regions (6,7) as well as information about the haplotype phasing, that is the combination of polymorphisms at the maternal and paternal alleles =-=(8)-=-. SNPs in intergenic and intronic elements like enhancers have been shown to regulate gene expression (9,10) and to contribute to human disorders (7,11). Recently, it was demonstrated that the activit...

Citation Context

...mbination of variations. The isogenicity of the flanking homologous sequences is an important issue. First, it could promote the targeting efficiency in human ES cells as was shown for mouse ES cells =-=(48,53)-=-. Second, bearing in mind that SNPs may influence transcription factor binding and gene expression (9,10), targeting with isogenic vectors should not disturb the existing genomic context. This will be...

Citation Context

...d using the HydroShear device (Digilab Genomic Solutions, MA, USA) and shearing assembly 4–40 kb (Zinsser Analytic, Frankfurt/Main, Germany) following the protocol for preparation of fosmid libraries =-=(35)-=-. The sheared DNA was end-repaired and ethanol precipitated according to the metagenomic DNA isolation protocol (Epicentre Biotechnologies). Fosmid library construction and DNA isolation from pools Fo...

Citation Context

... format that allows for rapid adaptation to functional analysis based on gene targeting (27,28) or transgenesis (30). A similar approach to isolate genomic regions in BACs has been published recently =-=(46)-=-. We use fosmids, because they are easy to handle, stable, suitable for genomic structural variation studies (2,5,22) and preparation of targeting constructs. Most importantly, compared to BAC librari...