The development of APE-PCR for the cloning of genomic insertion sites of DNA elements

Biologia ◽  
2013 ◽  
Vol 68 (4) ◽  
Author(s):  
Zhongjuan Xu ◽  
Yanli Li ◽  
Zhengwei Mao ◽  
Bin Yin
Keyword(s):  
Insertional Mutagenesis ◽  
Magnetic Particles ◽  
Magnetic Beads ◽  
Genetic Regulation ◽  
Insertion Site ◽  
Primer Extension ◽  
Successful Implementation ◽  
Nano Scale ◽  
Dna Elements ◽  
Insertion Sites

AbstractInsertional mutagenesis is a productive strategy for the exploration of genetic regulation of important biological and pathological processes, such as tumorigenesis. Successful implementation of this strategy depends heavily on an efficient approach to the identification of insertion sites present in the host genome. Here, we have introduced an easy and efficient protocol, called Adenosine-ended Primer Extension Polymerase Chain Reaction (APE-PCR), which represents several advantages, including the Addition technique we previously developed, primer extension approach coupled with biotin-streptavidin based purification, introduction of nano-scale magnetic particles, and digestion of DNA with a combination of enzymes. We have demonstrated that APE-PCR is able to amplify more and larger specific proviral insertion site (PIS)-derived fragments, with a lower non-specific background produced, fewer steps and less DNA samples required, flexibility in choice of restriction enzymes applied, at a lower cost. Replacement of regular magnetic beads with nano-scale ones in the protocol can further increase its power. Moreover, even with small amount of sample DNA, PISs can be recovered and analyzed. Thus, based on the results provided from this study, we believe that APE-PCR represents an efficient method in mapping of PISs and likely, the insertion sites of other types of DNA elements as well.

Automated Retroviral Insertion Site Sequence Analysis and Mapping Tool Followed by Database Analysis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5528-5528
Author(s):  
Stephanie Laufs ◽  
Frank A. Giordano ◽  
Agnes Hotz-Wagenblatt ◽  
Uwe Appelt ◽  
Daniel Lauterborn ◽  
...  
Keyword(s):  
Insertional Mutagenesis ◽  
Integration Site ◽  
Query Sequence ◽  
Cpg Islands ◽  
Insertion Site ◽  
High Throughput Analysis ◽  
Site Analysis ◽  
Conventional Analysis ◽  
Integration Site Analysis ◽  
Insertion Sites

Abstract Increasing use of retroviral vector-mediated gene transfer and recent reports on insertional mutagenesis in mice and humans created intense interest to characterize vector integrations on the genomic level. Techniques to determine insertion sites, mainly based on time consuming manual data processing and compilation, are thus commonly applied in gene therapy laboratories. Since a high variability in processing methods hampers further data comparison, there is an urgent need to systematically process the data arising from such analysis. The obtained sequences from the integration site analysis are judged to be authentic only if the matching part of the genomic query sequence is surrounded by the 5′LTR-sequence on the one side and the adapter-sequence on the other side. Therefore we developed an Integrationseq tool. In this task, different methods for converting the ABI sequence trace files to high quality sequences and for recognizing and deleting the LTR and adaptor parts of the isolated clones were implemented. If neither a primer nor a LTR could be found, the sequence is discarded. If the LTR is found on the complementary strand, the integration sequence is reversed. The remaining sequence between primer and LTR positions are taken as the n integration sequence and written to a sequence output file. We validated the Integrationseq tool using 259 trace files originating from integration site analysis (LM-PCR). Sequences can be trimmed by IntegrationSeq, leading to an increased yield of valid integration sequence detection, which has shown to be more sensitive (100%) than conventional analysis (94.3%) and 15 times faster than conventional analysis, while the specifities are equal (both 100%). Valid integration sequences get further processed with IntegrationMap for automatic genomic mapping. IntegrationMap runs 50 times faster than conventional methods and retrieves detailed information about whether integrations are located in or close to genes, the name of the gene, the exact localization in the transcriptional units and further parameters like the distance from the transcription start site to the integration. Further information, e.g. data about CpG-Islands, LINEs or SINEs, and their distances to the integration is also displayed. Output files generated by the task were found to be 99.8% identical with results retrieved by conventional mapping with the Ensembl alignment tool. Using both tools, IntegrationSeq and IntegrationMap, a validated, fast and standardized high-throughput analysis of insertion sites can be achieved for the first time.

scholarly journals Sequence and Insertion Sites of Murine Melanoma-Associated Retrovirus

1999 ◽  
Vol 73 (11) ◽  
pp. 9178-9186 ◽  
Author(s):  
Mengfeng Li ◽  
Xiaojun Huang ◽  
Zhenyu Zhu ◽  
Elieser Gorelik
Keyword(s):  
Insertional Mutagenesis ◽  
Insertion Site ◽  
Biological Significance ◽  
Melanoma Cells ◽  
Full Length ◽  
Noncoding Region ◽  
Chromosome 8 ◽  
Coding Region ◽  
Basic Leucine Zipper ◽  
Insertion Sites

ABSTRACT We previously showed that B16 melanoma cells produce ecotropic melanoma-associated retrovirus (MelARV) which encodes a melanoma-associated antigen recognized by MM2-9B6 monoclonal antibody. The biological significance of MelARV in melanoma formation remains unknown. We found that infection of normal melanocytes with MelARV resulted in malignant transformation. It is likely that MelARV emerged from the defective Emv-2 provirus, a single copy of ecotropic provirus existing in the genome of C57BL/6 mice. In the present study, we cloned and sequenced the full-length MelARV genome and its insertion sites and we completed sequencing of the Emv-2 provirus. Our data show that MelARV has a typical full-length retroviral genome with high homology (98.54%) to Emv-2, indicating a close relationship between both viruses. MelARV probably emerged as a result of recombination between Emv-2 and an endogenous nonecotropic provirus. Some observed differences in the gag and polregions of MelARV might account for the restoration of productivity and infectivity of a novel retrovirus that somatically emerged during melanoma formation. MelARV does not contain any oncogene and therefore might induce transformation by insertional mutagenesis. We sequenced two insertion sites of MelARV. The first insertion site represents the 3′ coding region of the c-maf proto-oncogene at 67.0 centimorgans (cM) on chromosome 8. The c-mafproto-oncogene encodes a basic leucine zipper protein homologous to c-fos and c-jun. Insertion of MelARV in BL6 melanoma cells resulted in the up-regulation of c-maf. It is noteworthy that the Emv-2 provirus is also inserted into a noncoding region at 61.0 cM on the same chromosome 8. The second insertion site is the 3′ noncoding region of the DNA polymerase gamma (PolG) gene on chromosome 7. The expression of PolG was not affected by the MelARV insertion. Further investigation of the biological significance of MelARV in melanoma formation is being undertaken.

Reduced Genotoxic Risk Using Foamy Viral Vectors for the Treatment of Canine Leukocyte Adhesion Deficiency

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3744-3744
Author(s):  
Thomas R. Bauer ◽  
Mehreen Hai ◽  
Rima L. Adler ◽  
James M. Allen ◽  
Laura M. Tuschong ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Insertional Mutagenesis ◽  
Leukocyte Adhesion ◽  
Severe Combined Immunodeficiency ◽  
Insertion Site ◽  
Leukocyte Adhesion Deficiency ◽  
Hematopoietic Stem ◽  
Insertion Sites ◽  
Immunodeficiency Disease

Abstract Gammaretroviral vectors used in recent gene therapy clinical trials have lead to several successes, such as in the treatment of X-linked severe combined immunodeficiency disease, but have also resulted in insertional activation of nearby oncogenes, leading to leukemia in four patients. We previously reported the successful treatment of four dogs with canine leukocyte adhesion deficiency (CLAD), a lethal genetic immunodeficiency disease caused by defects in the leukocyte integrin CD18, by transplanting foamy viral (FV) vector (deltaphiMscv-cCD18) - transduced, autologous CD34+ hematopoietic stem cells. To date, more than 2 years post transplant, all four dogs have maintained CD18+ leukocyte levels ranging between 5–10%, completely reversing of the CLAD phenotype, and have no clinical or laboratory evidence of hematological malignancy. To assess the potential genotoxicity of the FV gene therapy in the treatment of CLAD, we compared the insertion sites (ISs) found in the FV vector treated CLAD dogs with ISs found in CLAD dogs treated by gammaretroviral (RV) vectors (PG13/Mscv-cCD18). Insertion sites were identified by DNA sequence analysis of ligation-mediated PCR (LM-PCR) or linear amplification-mediated PCR (LAM-PCR) amplicons and subsequent comparison to the dog genome (canFam 2.0, May 2005). Insertion site analysis was performed for integrations that were in or within 50 kb of Refseq genes (using mouse/human orthologs). Analysis of the ISs revealed a reduced preference for FV vector integrations near transcription start sites compared to RV vector integrations (41% vs. 48%), fewer integrations near potential oncogenes (11% vs. 16%), and fewer integrations within genes in general (41% vs. 52%), in the FV vector treated animals compared to the RV vector treated animals. These clinically relevant data suggest that a reduced insertional mutagenesis potential exists when using FV vectors compared to RV vectors, and support the use of FV vectors in the treatment of human hematopoietic stem cell diseases such as LAD.

scholarly journals Tagmentation-Based Mapping (TagMap) of Mobile DNA Genomic Insertion Sites

2016 ◽  
Author(s):  
David L. Stern
Keyword(s):  
Insertion Site ◽  
Mobile Dna ◽  
Transposable Element Insertion ◽  
High Complexity ◽  
Rapid Preparation ◽  
Short Read Sequencing ◽  
The Past ◽  
Single Genome ◽  
Dna Elements ◽  
Insertion Sites

Multiple methods have been introduced over the past 30 years to identify the genomic insertion sites of transposable elements and other DNA elements that integrate into genomes. However, each of these methods suffer from limitations that can frustrate attempts to map multiple insertions in a single genome and to map insertions in genomes of high complexity that contain extensive repetitive DNA. I introduce a new method for transposon mapping that is simple to perform, can accurately map multiple insertions per genome, and generates long sequence “reads” that facilitate mapping to complex genomes. The method, called TagMap, for Tagmentation-based Mapping, relies on a modified Tn5 tagmentation protocol with a single tagmentation adaptor followed by PCR using primers specific to the tranposable element and the adaptor sequence. Several minor modifications to normal tagmentation reagents and protocols allow easy and rapid preparation of TagMap libraries. Short read sequencing starting from the adaptor sequence generates oriented reads that flank and are oriented toward the transposable element insertion site. The convergent orientation of adjacent reads at the insertion site allows straightforward prediction of the precise insertion site(s). A Linux shell script is provided to identify insertion sites from fastq files.

scholarly journals Large-scale discovery of mouse transgenic integration sites reveals frequent structural variation and insertional mutagenesis

2017 ◽  
Author(s):  
Leslie O. Goodwin ◽  
Erik Splinter ◽  
Tiffany L. Davis ◽  
Rachel Urban ◽  
Hao He ◽  
...  
Keyword(s):  
Large Scale ◽  
Structural Variation ◽  
Insertional Mutagenesis ◽  
Insertion Site ◽  
Essential Element ◽  
Mouse Genetics ◽  
High Rate ◽  
Dna Fragments ◽  
Structural Variations ◽  
Insertion Sites

ABSTRACTTransgenesis has been a mainstay of mouse genetics for over 30 years, providing numerous models of human disease and critical genetic tools in widespread use today. Generated through the random integration of DNA fragments into the host genome, transgenesis can lead to insertional mutagenesis if a coding gene or essential element is disrupted, and there is evidence that larger scale structural variation can accompany the integration. The insertion sites of only a tiny fraction of the thousands of transgenic lines in existence have been discovered and reported due in part to limitations in the discovery tools. Targeted Locus Amplification (TLA) provides a robust and efficient means to identify both the insertion site and content of transgenes through deep sequencing of genomic loci linked to specific known transgene cassettes. Here, we report the first large-scale analysis of transgene insertion sites from 40 highly used transgenic mouse lines. We show that the transgenes disrupt the coding sequence of endogenous genes in half of the lines, frequently involving large deletions and/or structural variations at the insertion site. Furthermore, we identify a number of unexpected sequences in some of the transgenes, including undocumented cassettes and contaminating DNA fragments. We demonstrate that these transgene insertions can have phenotypic consequences, which could confound certain experiments, emphasizing the need for careful attention to control strategies. Together, these data show that transgenic alleles display a high rate of potentially confounding genetic events, and highlight the need for careful characterization of each line to assure interpretable and reproducible experiments.

scholarly journals Tn5406, a New Staphylococcal Transposon Conferring Resistance to Streptogramin A and Related Compounds Including Dalfopristin

2002 ◽  
Vol 46 (8) ◽  
pp. 2337-2343 ◽  
Author(s):  
Julien Haroche ◽  
Jeanine Allignet ◽  
Névine El Solh
Keyword(s):  
Staphylococcus Aureus ◽  
Insertion Site ◽  
Single Copy ◽  
Amino Acid Identity ◽  
Plasmid Copy ◽  
Acid Identity ◽  
Abc Protein ◽  
Single Plasmid ◽  
Insertion Sites ◽  
Related Compounds

ABSTRACT We characterized a new transposon, Tn5406 (5,467 bp), in a clinical isolate of Staphylococcus aureus (BM3327). It carries a variant of vgaA, which encodes a putative ABC protein conferring resistance to streptogramin A but not to mixtures of streptogramins A and B. It also carries three putative genes, the products of which exhibit significant similarities (61 to 73% amino acid identity) to the three transposases of the staphylococcal transposon Tn554. Like Tn554, Tn5406 failed to generate target repeats. In BM3327, the single copy of Tn5406 was inserted into the chromosomal att554 site, which is the preferential insertion site of Tn554. In three other independent S. aureus clinical isolates, Tn5406 was either present as a single plasmid copy (BM3318), as two chromosomal copies (BM3252), or both in the chromosome and on a plasmid (BM3385). The Tn5406-carrying plasmids also contain two other genes, vgaB and vatB. The insertion sites of Tn5406 in BM3252 were studied: one copy was in att554, and one copy was in the additional SCCmec element. Amplification experiments revealed circular forms of Tn5406, indicating that this transposon might be active. To our knowledge, a transposon conferring resistance to streptogramin A and related compounds has not been previously described.

Magnetophoresis ‘meets’ viscoelasticity: deterministic separation of magnetic particles in a modular microfluidic device

Lab on a Chip ◽  
2015 ◽  
Vol 15 (8) ◽  
pp. 1912-1922 ◽  
Author(s):  
Francesco Del Giudice ◽  
Hojjat Madadi ◽  
Massimiliano M. Villone ◽  
Gaetano D'Avino ◽  
Angela M. Cusano ◽  
...  
Keyword(s):  
Microfluidic Device ◽  
Magnetic Particles ◽  
Magnetic Beads ◽  
Microfluidic Channel

Deflection of magnetic beads in a microfluidic channel can be improved through viscoelastic focusing.

A comparison of transparent polyurethane and dry gauze dressings for peripheral i.v. catheter sites: rates of phlebitis, infiltration, and dislodgment by patients

1997 ◽  
Vol 6 (5) ◽  
pp. 377-381 ◽  
Author(s):  
KA Tripepi-Bova ◽  
KD Woods ◽  
MC Loach
Keyword(s):  
Direct Observation ◽  
Meta Analysis ◽  
Insertion Site ◽  
Direct Visualization ◽  
Gauze Dressing ◽  
Insertion Sites ◽  
To Receive ◽  
Lower Frequencies

BACKGROUND: Before a meta-analysis by Hoffman et al was published, polyurethane dressings were used at insertion sites for peripheral i.v. catheters at our institution. On the basis of the results of the meta-analysis, we began to use gauze dressings. The change from polyurethane dressings to gauze dressings limited direct observation of the i.v. insertion site, and i.v. catheters were anecdotally reported not to be anchored as securely as before. OBJECTIVES: The purpose of this study was to compare the effects of the use of transparent polyurethane dressings and gauze dressings at insertion sites for peripheral i.v. catheters on the frequency of phlebitis, infiltration, and catheter dislodgment by patients. METHODS: Two hundred twenty-nine patients were randomized to receive either gauze (n = 121) or transparent polyurethane (n = 108) dressings, and observations were recorded. RESULTS: The frequency of catheter dislodgment by the patient was significantly higher (P < .05) in patients with the gauze dressing (15%) than in patients with the transparent polyurethane dressing (6%). A trend toward lower frequencies of phlebitis (1.8% vs 3.3%) and infiltration (17.6% vs 20.7%) was noted in the patients with the transparent polyurethane dressings. DISCUSSION: The clinical advantages of the transparent polyurethane dressings lie in the ease of direct visualization of the i.v. insertion site and the securement of the i.v. catheter. CONCLUSION: At our institution, given the decreased disruption of the i.v. therapy with the transparent polyurethane dressings and the lack of differences in the rates of phlebitis or infiltration with the two types of dressings, we prefer to use transparent polyurethane rather than gauze dressings at insertion sites for peripheral i.v. catheters.

Patency of radial arterial catheters

2001 ◽  
Vol 10 (2) ◽  
pp. 104-111 ◽  
Author(s):  
J Kaye ◽  
GR Heald ◽  
J Morton ◽  
T Weaver
Keyword(s):  
Sampling Methods ◽  
Insertion Site ◽  
Blood Sampling ◽  
Monitoring Systems ◽  
Site Location ◽  
Pressure Monitoring ◽  
Radiocarpal Joint ◽  
Background Data ◽  
Isotonic Sodium Chloride ◽  
Insertion Sites

BACKGROUND: Data on the influence of flush methods, blood-sampling methods, and site location on the patency of radial arterial catheters used for pressure monitoring are sparse. OBJECTIVES: To determine the effects of flush and blood-sampling methods, insertion site, and sex of patients on catheter patency. METHODS: In a randomized trial, 174 patients requiring radial arterial pressure monitoring were assigned to 4 groups: fast flush as needed and nonwaste blood sampling; fast flush as needed and waste blood sampling; fast flush every 4 hours and waste blood sampling; and fast flush every 4 hours and nonwaste blood sampling. All site locations were evaluated for patency, and all monitoring systems were maintained with isotonic sodium chloride solution. RESULTS: Nonpatent catheters were 4.23 times more likely in patients with insertion sites 3 cm or higher above the bend of the wrist than in patients with lower sites (P = .01). Duration of patency did not differ between catheters maintained with fast flush every 4 hours and those flushed as needed or between catheters according to the method of blood sampling. Women were 3.05 times more likely than men to have nonpatent catheters (P = .02). With insertion sites 3 cm or higher above the radiocarpal joint, nonpatency was 7.3 times more likely in women than in men (P < .001). CONCLUSIONS: Insertion sites closest to the bend of the wrist increase chances of maintaining patency. Catheters can be maintained with as-needed flushes, and either waste or nonwaste blood sampling can be used.

scholarly journals Continuous-Flow Separation of Magnetic Particles from Biofluids: How Does the Microdevice Geometry Determine the Separation Performance?

Sensors ◽  
2020 ◽  
Vol 20 (11) ◽  
pp. 3030 ◽  
Author(s):  
Cristina González Fernández ◽  
Jenifer Gómez Pastora ◽  
Arantza Basauri ◽  
Marcos Fallanza ◽  
Eugenio Bringas ◽  
...  
Keyword(s):  
Continuous Flow ◽  
Rational Design ◽  
Magnetic Particles ◽  
Magnetic Beads ◽  
Permanent Magnets ◽  
System Throughput ◽  
Separation Performance ◽  
High Particle ◽  
Section Shape ◽  
Geometrical Features

The use of functionalized magnetic particles for the detection or separation of multiple chemicals and biomolecules from biofluids continues to attract significant attention. After their incubation with the targeted substances, the beads can be magnetically recovered to perform analysis or diagnostic tests. Particle recovery with permanent magnets in continuous-flow microdevices has gathered great attention in the last decade due to the multiple advantages of microfluidics. As such, great efforts have been made to determine the magnetic and fluidic conditions for achieving complete particle capture; however, less attention has been paid to the effect of the channel geometry on the system performance, although it is key for designing systems that simultaneously provide high particle recovery and flow rates. Herein, we address the optimization of Y-Y-shaped microchannels, where magnetic beads are separated from blood and collected into a buffer stream by applying an external magnetic field. The influence of several geometrical features (namely cross section shape, thickness, length, and volume) on both bead recovery and system throughput is studied. For that purpose, we employ an experimentally validated Computational Fluid Dynamics (CFD) numerical model that considers the dominant forces acting on the beads during separation. Our results indicate that rectangular, long devices display the best performance as they deliver high particle recovery and high throughput. Thus, this methodology could be applied to the rational design of lab-on-a-chip devices for any magnetically driven purification, enrichment or isolation.

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