生物化学技术

To construct the recombinant adenovirus vector containing the cDNA for recombinant mouse cyclic nucleotide phosphodiesterase 3B (mPDE3B), the cDNA for mPDE3B was subcloned into pACCMV.pLpA. Subsequently, this recombinant plasmid, pACCMV.mPDE3B, was cotransfected with pJ ...

In many instances, pulsed-field gel electrophoresis (PFGE) is used to construct a map of a large region from the human or mouse genome. Sometimes, a gene or a gene-complex are very large, as in the case of the Duchenne muscular dystrophy gene (1) or the major histocompatibility complex (MHC) (2,3). Freque ...

Contour-clamped homogeneous electric field (CHEF) gelelectrophoresis is a particular formulation of pulsed-field gel electrophoresis (PFGE), which uses an array of electrodes positioned around the gel (on a contour) and clamped to specific voltages to produce a nearly homogene ...

Among the techniques to separate large DNA fragments, field-inversion gel electrophoresis (FIGE, 1)is probably the easiest to perform with a minimum of special equipment. Indeed, the only requirement besides a regular gel electrophoresis box and a power supply is a device enabling the per ...

Some types of pulsed-field gel electrophoresis (PFGE), including CHEF (contour-clamped homogeneous electric field), give straight lanes and excellent separations of a wide range of DNA sizes on one gel (ref. 1 and Chapters 1,2,5–7). Here conditions are described for the separation of DNA in the ...

The methods discussed in this chapter were the first pulsed-field gel electrophoresis (PFGE) methods developed. As a curiosity, in the first publications, PFGE stood for pulsed-field gradient gel electrophoresis (1). However, later it was recognized that field gradients or inhomoge ...

Rotating field gel electrophoresis (ROFE) (1–4) is an alternative to orthogonal field alternating (OFAGE) (5), transverse alternating field (TAFE) (6), field inversion (FIGE) (7), or related pulsed-field gel electrophoretic procedures for the separation of very large nucleic acid m ...

Transverse alternating-field electrophoresis (TAFE) refers to a pulsed-field system that uses a vertical gel and a simple electrode geometry. A schematrc of the apparatus is shown in Fig. 1 (1). The electrophoresis tank is a large plexiglass box, in which the gel stands vertically, supported at ...

The preparation and manipulation of very large DNA molecules for pulsed-field gel electrophoresis (PFGE) requires more care than normally used for smaller molecules. The general protocol used is the preparation of DNA directly in solid agarose blocks (plugs) or beads. Intact cells are en ...

A prerequisite for physical mapping by pulsed-field gel electrophoresis (PFGE) is the saturation of a given genome or chromosomal region with single copy markers. One possible way to achieve this goal is by construction of a saturated restriction fragment length polymorphism (RFLP) map. ...

This novel way of preparing chromosomes for pulsed-field gel electrophoresis (PFGE) takes advantage of the fact that the whole chromosome population is synchronized in metaphase. This is a very important step toward their intact separation by PFGE; for instance, a standard preparation ...

Pulsed-field gel electrophoresis (PFGE) has allowed the resolution of very large DNA fragments from any organism. To apply PFGE to practical problems, such as genetic mapping and map-based gene cloning, it is necessary to specifically generate large DNA fragments that can then be separated ...

The multisample slab gels used for pulsed-field gel electrophoresis (PFGE) can produce gel patterns of hundreds of samples per day (see, for example, ref. 1). Because of this efficiency in the production of data, the rate-limiting steps in obtaining information at times are analysis, organiza ...

The aim of this chapter is to provide a very practical set of instructions that will enable workers with some experience in standard DNA preparation and electrophoresis techniques to prepare and analyze mammalian DNA by pulsed-field gel electrophoresis (PFGE; see also refs. 1 and 2 for additi ...

For the analysis of large DNA fragments using pulsed-field gel electrophoresis (PFGE), it is necessary to first embed the cells in agarose to prevent shearing of the DNA during protein extraction and restriction enzyme digestion. The speed and efficiency with which these steps are performed ...

Two-dimensional pulsed-field gel electrophoresis (2D-PFGE) is a powerful PFGE technique for the restriction mapping of bacterial genomic DNA. The method consists of two sequential steps of restriction endonuclease digestion and separation of the fragments by PFGE: 1. Step A, in which p ...

The technology of cloning large fragments of DNA in yeast as yeast artificial chromosomes (YACs) (1), combined with that of electro-phoretic separation of large fragments by pulsed-field gel electrophoresis (PFGE), has revolutionized research in molecular genetics. In addition to t ...

Yeast artificial chromosomes (YACs; 1) are rapidly becoming the major cloning system to study eukaryotic genomes by physical mapping and chromosome walking projects (1–6). The main advantages of YACs over prokaryotic-based cloning systems are their large insert capacity (100–1500 ...

Protozoa represent a diverse group of single-celled eukaryotes, many of which have parasitic life styles, infecting hundreds of millions of people. Unique aspects of their biology relate to their distinct evolutionary position and their complex life cycles, frequently involving d ...

Long-term and stable hepatocyte culture systems have a wide variety of uses, both in basic science and in the development of hepatocyte-based applications. In most cases, long-term cultures of hepatocytes are superior to traditional cultures in collagen-coated dishes, which only tran ...