细胞功能测定

Oligonucleotide-directed in vitro mutagenesis is an established tool for the investigation of protein structure and function, and increasingly for studying the role of DNA sequences in gene expression. The method described here is based on the phosphorothioate technique develo ...

Site-directed mutagenesis has become an extremely powerful means by which the nature and function of critical amino acids within a protein can be assessed and altered. However, the ability to exploit this technique requires that detailed structural information about the protein (or re ...

Oligonucleotide-directed mutagenesis has become the primary method for testing theories of protein structure/function and control of gene expression by specifically altering DNA sequences. Random oligonucleotides can be used to extend this approach to genes where less stru ...

Randomly induced point mutations provide an effective strategy to recognize and characterize functionally significant features of cloned DNAs. When coupled with sensitive assays for gene function, random mutagenesis permits one to resolve active DNA motifs within a gene’s regu ...

Generating random mutations in a DNA fragment of a specific size is often the method of choice for changing specific properties of an encoded protein or for studying the functional properties of a specific DNA fragment. In all these cases it is helpful and in some cases essential to have a screening met ...

Classically, studies on the relationships between structure and function have focused on the synthesis of individual molecular species and then analyzed their functions in cells. Recently, an entirely different approach has become feasible. A series of techniques has made it possib ...

Polymerase chain reaction (PCR) is a powerful technique for the amplification of specific DNA sequences, for the generation of chimeric molecules, and for site-directed mutagenesis. It allows the rapid generation of relatively large quantities of mutated DNA. Drawbacks are the costs of ...

Saturation mutagenesis is one approach for determining the contributions of individual base pairs or amino acids to the structure and function of defined DNA sequence elements or proteins, respectively (1). If a simple phenotypic screen is available, saturation mutagenesis can be used ...

Cassette mutagenesis methods that introduce site-specific sequence changes into a target gene are powerful tools for the manipulation of proteins to analyze then structure and function (1–3). Typically, a small target region is excised by cleavage at two constructed or naturally occu ...

In Chapter 25 we described linker scanning mutagenesis by oligonucleotide ligation. In this chapter we describe a more recent and versatile procedure that makes use of amplification by the polymerase chain reaction (PCR). For a description of the traditional method of generating linker ...

The purpose of linker-scanning mutagenesis is to create a series of mutant molecules in which individual sections are sequentially replaced with the same “neutral” mutant sequence (see Fig. 1). Typically the technique has been applied to DNA and has most commonly been used to determine the cis- ...

Site-directed mutagenesis techniques allow selective engineering of gene sequences. In in vitro site-directed mutagenesis, base alterations are introduced into a target sequence by incorporating DNA base changes within a primer utilized in the DNA synthesis step. Several site- ...

Site-directed mutagenesis is an important component of modern molecular biology. The use of this technique became necessary both in our studies of promoter function and in the analysis of protein structure-function relationships. Although several polymerase chain reaction (PC ...

Site-directed mutagenesis is a powerful tool used in modern molecular biology for protein and genetic engineering. A number of simple and elegant protocols are available to introduce mutations into a target DNA sequence. However, there are only a few methods described for deletion mutage ...

Site-directed mutagenesis is one of the fundamental tools available to probe the structure and function of proteins and cellular controlling mechanisms. Conventionally, site-directed mutagenesis methods can be reduced to two steps: strand separation and annealing of a mutagen ...

We present in this chapter a polymerase chain reaction (PCR)-based method to simultaneously introduce and remove large fragments of DNA in a single mutagenesis reaction without the need for restriction sites. We have favored the use of long single-stranded DNA primers synthesized by asym ...

Using polymerase chain reaction (PCR) it is possible to amplify a segment of DNA by a factor of approx 220 under standard conditions (1). Any mutations present in the oligonucleotides used to prime the polymerization reactions will be incorporated in the resulting PCR products. Thus, a specific ...

Among the various mutagenesis procedures based on polymerase chain reaction (PCR), the “megaprimer” method appears to be the simplest and most versatile. The method utilizes three oligonucleotide primers and two rounds of PCR performed on a DNA template containing the cloned gene to be mut ...

Polymerase chain reaction (PCR) is a powerful and efficient method allowing enzymatic amplification of small quantities of DNA. This technology has also been widely used as a quick and efficient alternative for introducing mutations into specific DNA sequences. Various general met ...

Site-directed mutagenesis and the polymerase chain reaction (PCR) represent two powerful techniques that have led to rapid advances in our understanding of gene expression and function. Early protocols for site-directed mutagenesis depended on the production of single-stran ...