遗传学实验技术

Reporter genes provide easy and efficient methods for the indirect measurement of relative rates of transcription. Utilizing common DNA cloning methods, a putative regulatory region can be coupled to the coding sequence of a reporter gene such that expression of the reporter protein pro ...

The term “liposome” originated approximately 30 years ago to refer to lipid bilayers that form colloidal particles in an aqueous medium (1). Artificially developed liposomes were used effectively by 1980 to deliver DNA to cultured cells (2). The next advancement in liposome delivery vehi ...

DEAE-dextran transfection is one of the oldest chemical, nonviral methods developed to transfer RNA or DNA to cultured mammalian cells (1,2). Early transfection studies used viral RNA (1) and DNA (2), which were easy to propagate and purify, and allowed phenotypic discrimination of the trans ...

Calcium phosphate and DEAE-dextran reagents were incorporated into the first chemical methods that successfully transferred nucleic acid directly to cultured mammalian cells in a process referred to as transfection (1–4). Early transfection studies used viral RNA (1) and DNA (2,4), w ...

The analysis of chromatin structure at single-nucleotide resolution (genomic footprinting) has long been considered technically difficult, at least in mammalian cells. Recently, techniques have been developed that give a sufficient specificity and sensitivity to analyze s ...

Electroporation is a process in which a controlled electrical pulse is applied to cells, inducing a transient destabilization of the cell membrane. During this time, the cells are highly permeable to exogenous substances in the surrounding media. DNA, proteins, and small molecules are all t ...

Research in molecular genetic studies and in transgenic biotechnology has resulted in excellent advancements in exploiting gene transfer techniques. Basic research in transcriptional and/or translational control, protein engineering, cellular immunological mecha ...

Regulation of gene expression is controlled through the combinatorial action of multiple transcription factors, which function to activate or repress transcription via binding to cis-regulatory elements. Such regulatory elements are usually present in the promoter sequen ...

Variation coupled to selection is the hallmark of natural evolution. Although there is no full agreement concerning the best way to create variation, mutation or recombination (1), computational simulation studies have demonstrated the importance of homologous recombination in ...

Family shuffling, which generates chimeric progeny genes by recombining a set of naturally occurring homologous genes, is an extremely powerful approach for in vitro protein evolution. In comparison with other in vitro protein evolution methods, family shuffling has the advantage of ...

DNA shuffling is a method for in vitro recombination of homologous genes invented by W.P.C Stemmer (1). The genes to be recombined are randomly fragmented by DNaseI, and fragments of the desired size are purified from an agarose gel. These fragments are then reassembled using cycles of denaturati ...

Whole genes, plasmids, and even viral genomes can be assembled from relatively short, synthetic, overlapping oligodeoxyribonucleotides (oligos) by DNA polymerase extension (1,2). Once the initial investment in a set of oligos spanning the length of a gene has been made, a mutation can be gen ...

The conventional method for cloning a DNA fragment is to insert it into a vector and ligate it (1). Although this method is commonly used, it is labor intensive because the ratio and concentrations of the DNA insert and the vector must be optimized. Even then, the resulting library is often plagued with unw ...

Sequence mapping of combinatorial libraries is of interest for evaluation of library diversity and homogeneity as well as for bias detection and analysis. This is particularly useful for library improvement by robotic equalization. Macro- and microarray based experimental proc ...

Sequence mapping consists in the characterization of a set of sequence segments in a library of related genes or PCR products. This procedure can be applied to analyze natural sequence variants, for example allelic distribution of human genomic sequences for disease, drug metabolism pre ...

In vitro recombination is often used to generate genetic diversity for directed evolution. Recombination techniques that rely on fragment hybridization yield libraries with preferred crossover positions and, in some cases, a bias toward incorporation of one parent over the others. ...

High recombination frequency and ease of manipulation made the budding yeast, Saccharomyces cerevisiae, the model eukaryotic organism for studies on homologous recombination (1,2). Mutagenesis of intrinsic genes (3) and the construction of vectors (4) are applications of in vivo r ...

Directed evolution usually starts from the analysis of variant genes. Among methods used to produce such diversity, recombination between members of a gene family is a frequent approach (1). Building a library of mosaic genes can been achieved by fragmentation of parental genes and subsequ ...

SCRATCHY is a combination of the incremental truncation for the creation of hybrid enzymes (ITCHY) technology (1) and DNA shuffling (2). It generates combinatorial libraries of hybrid proteins consisting of multiple fragments from two or more parental DNA sequences with no restriction ...

Incremental truncation is a method for creating a library of every one base truncation of dsDNA. Incremental truncation libraries can be created by time dependent Exo III digestions (1) or by the incorporation of α-phosphorothioate dNTPs (αS-dNTPs) (2). The fusion of two incremental trunc ...