基因表达差异显示实验

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 too ...

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 gene ...

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–D ...

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 expe ...

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 reco ...

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 ex ...

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 t ...

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 target ...

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 L ...

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, fluore ...

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 sing ...

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- ...

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 subs ...

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- ...

Uracil excision-based cloning through USER™ (Uracil-Specific Excision Reagent) is an efficient ligase-free cloning technique that comprises USER cloning, USER fusion, and USER cassette-free (UCF) USER fusion. These USER-derived cloning techniques enable seamless assemb ...

The emerging field of synthetic biology requires novel cloning techniques that allow the rapid assembly of multiple expression units to build artificial genetic circuits. Here, we describe a rapid, flexible, and cost-efficient cloning method that requires only standard laborato ...

Identification of unknown sequences that flank known sequences of interest requires PCR amplification of DNA fragments that contain the junction between the known and unknown flanking sequences. Since amplified products often contain a mixture of specific and nonspecific prod ...

Sequence and ligation-independent cloning (Nat Methods 4:251–256, 2007) is a powerful tool for the construction of multi-fragment complex plasmids in a simple and efficient manner. Plasmids consisting of 6–7 DNA fragments can be assembled in a single day, with additional 2 days for screeni ...

The BioBrick idea was developed to introduce the engineering principles of abstraction and standardization into synthetic biology. BioBricks are DNA sequences that serve a defined biological function and can be readily assembled with any other BioBrick parts to create new BioBric ...

This chapter introduces the software FastPCR as an integrated tools environment for PCR primer and probe design. It also predicts oligonucleotide properties based on experimental studies of PCR efficiency. The software provides comprehensive facilities for designing primers ...