细胞技术

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 ...

The polymerase chain reaction (PCR) (1,2) is now a fundamental tool of molecular biology. Although PCR provides the basis for a variety of sensitive analytical techniques, it can also be used in a synthetic capacity to generate large quantities of specific DNA fragments. The alteration of ampli ...

Protocols for site-directed mutagenesis are widely used in molecular biology and include many polymerase chain reaction (PCR)-based methods that have been developed in order to achieve efficient mutagenesis of a target DNA sequence (1–4). However, some of these methods described req ...

Before the advent of polymerase chain reaction (PCR) technology, many methods for site-directed mutagenesis basically relied on enzymatic extension of a mutagenic oligonucleotide annealed to a single-stranded template and amplification of the ligase-sealed double-stran ...

An important manipulation in molecular genetics is to make ordered deletions into a cloned piece of DNA. The most widely used application of this method is in DNA sequencing. Ordered deletions can also be used in delineating sequences that are important for the function of a gene, such as those requi ...

In vitro site-directed mutagenesis has been widely used in vector modification, and in gene and protein structure/function studies (1,2). This procedure typically employs one or more oligonuleotides to introduce defined mutations into a DNA target of known sequence (2–9). A variation of ...

The generation of nested deletions within cloned fragments of DNA has important applications in molecular biology. For DNA sequencing, nested deletions provide a series of overlapping templates that can be used to generate a composite sequence with a single sequencing primer; in gene and ...

Oligonucleotides can bind in the major groove of duplex DNA and form triple helices in a sequence-specific manner 1–4). Progress in elucidating the third strand binding code has raised the possibility of developing nucleic acids as sequence-specific reagents for research and possibly c ...

A number protocols have been established for site-directed mutagenesis based on the work of Smith (1) and Hutchinson et al. (2) that use hybridization of a mismatched oligonucleotide to a DNA template followed by second-strand synthesis by a DNA polymerase. These techniques provide effici ...

For convenience the term quiescent cells will be used in this chapter to define normal or tumor cells, untreated by any specific drugs, that do not progress through the mitotic cycle for an extended period of time. The definition is purposefully wide to cover a variety of situations, often with differ ...

Analysis of the cell cycle was significantly advanced by the development of photometric methods for quantitative measurement of biochemical constituents in single cells. Population biochemical analysis could then be performed on a cell-by-cell basis, and distinct subpopulat ...

The dispersal of solid tissues into a single-cell suspension has become an integral component of many current techniques for quantitative cytokinetic analysis of both normal and neoplastic tissues. This is particularly evident in the application of flow cytometric (FCM) procedur ...

The measurement of percent labeled mitoses (PLM, or alternatively, FLM, for fraction of labeled mitoses) was developed in 1959 by Quastler and Sherman (8) for estimating the durations of the component phases of the cell cycle.