Springer protocols feed by Genetics/Genomics 
This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.
This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.PCR-Based Bioprospecting for Homing Endonucleases in Fungal Mitochondrial rRNA Genes
Fungal mitochondrial genomes act as “reservoirs” for homing endonucleases. These enzymes with their DNA site-specific cleavage activities are attractive tools for genome editing and gene therapy applications. Bioprospecting and characterization of naturally occurring homing endonucleases offers an alternative to synthesizing artificial endonucleases. Here, we describe methods for PCR-based screening of fungal mitochondrial rRNA genes for homing endonuclease encoding sequences, and we also provide protocols for the purification and biochemical characterization of putative native homing endonucleases. (Source: Sp...
Source: Springer protocols feed by Genetics/Genomics - February 13, 2014 Category: Genetics & Stem Cells Source Type: news
Bioinformatic Identification of Homing Endonucleases and Their Target Sites
Homing endonuclease genes (HEGs) are a large, phylogenetically diverse superfamily of enzymes with high specificity for especially long target sites. The public genomic sequence databases contain thousands of HEGs. This is a large and diverse arsenal of potential genome editing tools. To make use of this natural resource, one needs to identify candidate HEGs. Due to their special relationship with a host gene, it is also possible to predict their cognate target sequences. Here I describe the HomeBase algorithm that was developed to this end. A detailed description of the computational pipeline is provided with emphasis on ...
Source: Springer protocols feed by Genetics/Genomics - February 13, 2014 Category: Genetics & Stem Cells Source Type: news
Homing Endonucleases: From Genetic Anomalies to Programmable Genomic Clippers
Homing endonucleases are strong drivers of genetic exchange and horizontal transfer of both their own genes and their local genetic environment. The mechanisms that govern the function and evolution of these genetic oddities have been well documented over the past few decades at the genetic, biochemical, and structural levels. This wealth of information has led to the manipulation and reprogramming of the endonucleases and to their exploitation in genome editing for use as therapeutic agents, for insect vector control and in agriculture. In this chapter we summarize the molecular properties of homing endonucleases and disc...
Source: Springer protocols feed by Genetics/Genomics - February 13, 2014 Category: Genetics & Stem Cells Source Type: news
Redesigning the Specificity of Protein–DNA Interactions with Rosetta
Building protein tools that can selectively bind or cleave specific DNA sequences requires efficient technologies for modifying protein–DNA interactions. Computational design is one method for accomplishing this goal. In this chapter, we present the current state of protein–DNA interface design with the Rosetta macromolecular modeling program. The LAGLIDADG endonuclease family of DNA-cleaving enzymes, under study as potential gene therapy reagents, has been the main testing ground for these in silico protocols. At this time, the computational methods are most useful for designing endonuclease variants that can ...
Source: Springer protocols feed by Genetics/Genomics - February 13, 2014 Category: Genetics & Stem Cells Source Type: news
Identification and Analysis of Genomic Homing Endonuclease Target Sites
Homing endonucleases (HEs) are highly site-specific enzymes that enable genome engineering by introducing DNA double-strand breaks (DSB) in genomic target sites. DSB repair from an HE-induced DSB can promote target site gene deletion, mutation, or gene addition, depending on the experimental protocol. In this chapter we outline how to identify potential genomic target sites for HEs with known target site specificities and the different experimental strategies that can be used to assess site cleavage in living cells. As an example of this approach, we identify potential human genomic target sites for the LAGLIDADG HE I-CreI...
Source: Springer protocols feed by Genetics/Genomics - February 13, 2014 Category: Genetics & Stem Cells Source Type: news
Bioinformatics Identification of Coevolving Residues
Positions in a protein are thought to coevolve to maintain important structural and functional interactions over evolutionary time. The detection of putative coevolving positions can provide important new insights into a protein family in the same way that knowledge is gained by recognizing evolutionarily conserved characters and characteristics. Putatively coevolving positions can be detected with statistical methods that identify covarying positions. However, positions in protein alignments can covary for many other reasons than coevolution; thus, it is crucial to create high-quality multiple sequence alignments for coev...
Source: Springer protocols feed by Genetics/Genomics - February 13, 2014 Category: Genetics & Stem Cells Source Type: news
Engineering and Flow-Cytometric Analysis of Chimeric LAGLIDADG Homing Endonucleases from Homologous I-OnuI-Family Enzymes
LAGLIDADG homing endonucleases (LHEs) are valuable tools for genome engineering, and our ability to alter LHE target site specificity is rapidly evolving. However, widespread use of these enzymes is limited due to the small number of available engineering scaffolds, each requiring extensive redesign to target widely varying DNA sequences. Here, we describe a technique for the chimerization of homologous I-OnuI family LHEs. Chimerization greatly expands the pool of unique starting scaffolds, thereby enabling more effective and efficient LHE redesign. I-OnuI family enzymes are divided into N- and C-terminal halves based on s...
Source: Springer protocols feed by Genetics/Genomics - February 13, 2014 Category: Genetics & Stem Cells Source Type: news
Homing Endonuclease Target Determination Using SELEX Adapted for Yeast Surface Display
Knowing the target sequence of a DNA-binding protein is vital in obtaining fundamental characteristics of the protein and evaluating properties of the protein–DNA interaction. For example, novel homing endonucleases cannot be proven to be functional until a predicted target site is tested. Unfortunately, target site prediction is not always easy, or even possible, depending on the amount of sequence data available. Here we describe a modification of SELEX using yeast surface display that can quickly and inexpensively resolve DNA-binding targets in high throughput for proteins without any prior assumptions or knowledg...
Source: Springer protocols feed by Genetics/Genomics - February 13, 2014 Category: Genetics & Stem Cells Source Type: news
Homing Endonuclease Target Site Specificity Defined by Sequential Enrichment and Next-Generation Sequencing of Highly Complex Target Site Libraries
Homing endonucleases (HEs) are DNA sequence-specific enzymes that recognize and cleave long target sites (14–40 bp) to generate double-strand breaks (DSBs). Their high site recognition specificity and tight coupling of binding and cleavage make HEs attractive reagents for targeted genome manipulation. In order to delineate the target site specificity of HEs and facilitate HE engineering, we have developed a method for comprehensive target site profiling of HEs cleavage specificity using partially randomized target site libraries and high-throughput DNA sequencing. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - February 13, 2014 Category: Genetics & Stem Cells Source Type: news
Quantifying the Information Content of Homing Endonuclease Target Sites by Single Base Pair Profiling
Homing endonucleases (HEs) are native proteins that recognize long DNA sequences with high site specificity in vitro and in vivo. The target site specificity of HEs is high, though not absolute. For example, members of the well-characterized LAGLIDADG family of homing endonucleases (the LHEs) recognize target sites of ~20 base pairs, and can tolerate some target site base pair changes without losing site binding or cleavage activity. This modest degree of target site degeneracy is practically useful once defined and can facilitate the engineering of LHE variants with new DNA recognition specificities. In this chapter, we o...
Source: Springer protocols feed by Genetics/Genomics - February 13, 2014 Category: Genetics & Stem Cells Source Type: news
Rapid Determination of Homing Endonuclease DNA Binding Specificity Profile
Evaluating the binding specificity and identifying the most preferred target sequence for a homing endonuclease often represents a key step during its characterization, engineering, and application for genome engineering. This chapter describes a high-throughput, fluorescence-based, competition-binding assay which can be used to measure the relative binding affinities of the homing endonuclease to a large number of DNA target site variants in a single experiment. The base preference at each position of the target sequence can be quantitated based on this assay, and the overall binding specificity of the enzyme can thereby ...
Source: Springer protocols feed by Genetics/Genomics - February 13, 2014 Category: Genetics & Stem Cells Source Type: news
Golden Gate Cloning
DNA assembly methods are essential tools for biological research and biotechnology. Therefore various methods have been developed to clone DNA fragments of interest. Conventional methods usually require several cloning steps to generate a construct of interest. At each step, a single DNA fragment is transferred from a donor plasmid or PCR product to a recipient vector. In the past few years, a number of methods have been developed to facilitate and speed up this process. One of these methods, Golden Gate cloning, allows assembling up to nine fragments at a time in a recipient plasmid. Cloning is performed by pipetting in a...
Source: Springer protocols feed by Genetics/Genomics - January 11, 2014 Category: Genetics & Stem Cells Source Type: news
Circular Polymerase Extension Cloning
High-throughput genomics, proteomics, and the emerging field of synthetic biology demand ever more convenient, economical, and efficient technologies to assemble and clone genes, gene libraries, and synthetic pathways. Here, we describe an extremely simple, efficient, and cost-effective cloning method, circular polymerase extension cloning (CPEC), for complex, combinatorial, or multi-fragment assembly as well as routine cloning. This method uses a single polymerase to assemble and clone multiple inserts with any vector in a one-step reaction in vitro. No restriction digestion, ligation, or single-stranded homologous recomb...
Source: Springer protocols feed by Genetics/Genomics - January 11, 2014 Category: Genetics & Stem Cells Source Type: news
A Single-Tube Assembly of DNA Using the Transfer-PCR (TPCR) Platform
DNA cloning is a basic methodology employed for multiple applications in all life-science disciplines. In order to facilitate DNA cloning we developed Transfer-PCR (TPCR), a novel approach that integrates in a single tube, PCR amplification of the target DNA from an origin vector and its subsequent integration into the destination vector. TPCR can be applied for incorporation of DNA fragments into any desired position within a circular plasmid without the need for purification of the intermediate PCR product and without the use of any commercial kit. TPCR reaction is most efficient within a narrow range of primer concentra...
Source: Springer protocols feed by Genetics/Genomics - January 11, 2014 Category: Genetics & Stem Cells Source Type: news
Application of the Restriction-Free (RF) Cloning for Multicomponents Assembly
Molecular manipulations, including DNA cloning and mutagenesis, are currently employed on a routine basis in all life science disciplines. Over the last decade new methodologies have emerged that expanded and facilitated the applications for DNA cloning. The classical Ligation-Dependent Cloning (LDC) is gradually replaced by Ligation-Independent Cloning (LIC) techniques. The Restriction-Free (RF) cloning was originally developed for introduction of a foreign DNA into a plasmid at any desired position. The RF methodology is based on generation of a PCR product, which serves as a set of mega-primers for subsequent incorporat...
Source: Springer protocols feed by Genetics/Genomics - January 11, 2014 Category: Genetics & Stem Cells Source Type: news
