遗传学实验技术

DNA shuffling is a developed technique that allows accelerated and directed protein evolution in vitro. In this method, the acquisition of genes encoding improved proteins is done in two steps: First, a single gene is mutagenized, and desired mutant genes are selected; second, the mutant genes ...

Mutagenesis is an essential process in evolution, generating an array of genetically unique organisms from which natural selection selects the best-suited to the immediate environment. Compatible and favorable mutations quickly converge; because offspring may inherit dist ...

In vitro protein evolution is an efficient approach to study the structure and function of a protein, or to enhance its industrial utility (1). One round of evolution consists of random mutation of a protein-coding gene, expression the resulting library in a population of micro-organisms, and h ...

Site-directed mutagenesis (SDM) is a powerful tool for the study of gene expression/regulation and protein structure and function. Hutchinson et al. (1) developed a general method for the introduction of specific changes in DNA sequence, which involves hybridization of a synthetic oli ...

Polymerase chain reaction (PCR)-based site-directed mutagenesis (SDM) methods are widely used in molecular biology to selectively alter gene sequences (1). However, common protocols allow for the introduction of only one (1–3) or two (4) independent point mutations at a time, followed by ...

Oligonucleotide-based, site-directed mutagenesis (SDM) of cloned DNA has become a fundamental tool of modern molecular biology, used to introduce insertions, deletions, and substitutions into DNA. Current techniques available for performing in vitro mutagenesis fall into th ...

Since the development of the polymerase chain reaction (PCR) technique (1), its potential use as a tool for site-directed mutagenesis (SDM) has been extensively explored, as illustrated by several chapters in this volume. In particular, the relative ease with which DNA fragments of 2–3 kb can be g ...

The invention of polymerase chain reaction (PCR) (1,2) provided a powerful tool to modify DNA sequences in genetic engineering. With numerous mutagenesis methods available, such as traditional sequential PCR (3), “megaprimer PCR”(4–7), marker-coupled PCR (8), and so on, introducing cha ...

The invention of site-directed amino acid (AA) mutagenesis (1) has revolutionized the way in which enzyme mechanism and specificity can be probed, while simultaneously providing a route for engineering novel enzyme properties. Prior to this very precise method of AA replacement, prote ...

Site-directed mutagenesis (SDM) by deletion of a unique restriction site, introduced by Deng and Nickoloff (1), allows site-specific mutagenesis of a plasmid DNA without any subcloning steps. This procedure uses two mutagenic primers: one carries the desired mutation; the second, acti ...

During the past decade, a number of methods using polymerase chain reaction (PCR) for the generation of specific mutations in any nucleotide sequence have been described, these methods include such drawbacks as the possibility of generating undesired secondary mutations, because of t ...

Olfactory receptors are a specialized set of receptor cells responsible for the detection of odors. These cells are G protein-coupled receptors and expressed in the cell membranes of olfactory sensory neurons. Once a cell is activated by a ligand, it initiates a signal transduction cascade t ...

Olfactory receptors (ORs) are expressed at the cell surfaces of olfactory sensory neurons lining the olfactory epithelium and are the first actors for the perception and recognition of odorants. The olfactory system is mostly activated by ORs and odorants in the cAMP pathway. To date, few human ...

Odor detection and discrimination by olfactory systems in vertebrates and invertebrates depend both on the selective expression of individual olfactory receptor genes in subpopulations of olfactory sensory neurons, and on the targeting of the encoded proteins to the exposed, cil ...

Mammalian species have evolved a large and diverse number of odorant receptors (ORs). These proteins comprise the largest family of G-protein-coupled receptors (GPCRs) known, amounting to ∼1,000 �different receptors in the rodent. From the perspective of olfactory coding, the avail ...

The large number of olfactory receptors (ORs) expressed by various mammalian and insect species, as well as the large number of potential odorant ligands, has made the pairing of odorants with receptors �(de-orphaning) exceedingly difficult. These efforts are further complicated by di ...

Insects rely significantly on olfactory cues for recognition and finding of vital resources such as food and mates. Odor detection is mediated by primary sensory neurons housed in individualized cuticular structures, the sensilla. Using microelectrode-based techniques, it is po ...

Mammals have between 400 and 1,300 functional odorant receptor (OR) genes in their genomes. Each olfactory sensory neuron in the nose expresses only one single type of OR out of this vast repertoire. The OR expressed by an olfactory sensory neuron determines its functional activity and wiring to t ...

Cell surface expression of recombinant olfactory receptors (ORs) is a major limitation in characterizing their functional nature. We have shown that the recombinant expression of a human OR, OR 17-210, in the baculovirus/Sf9 insect cell system allows this protein to be expressed at the cell s ...

The first bottleneck in olfactory receptor (OR) studies is producing sufficient quantities of soluble, �functional, and stable receptors. Commercial cell-free in vitro translation systems can be used to produce milligrams of soluble and functional receptors within several hou ...