生物化学技术

Advances in DNA sequencing techniques have made it possible to routinely sequence long DNA molecules (1,2). Recently double-stranded DNA has been directly sequenced with chain terminators (3), and this eliminates the tedious task of subcloning the fragments into single-stranded M13 ...

DNA sequencing is a technique that has become central to many laboratories engaged in molecular biology. The two basic sequencing methods commonly used are known as the Maxam-Gilbert method of chemical cleavage (1) and the dideoxy chain termination method developed originally by Sanger ...

For the majority of purposes, such as restriction mapping and sizing of cloned fragments, electrophoresis of DNA in horizontal agarose gels is perfectly adequate. As the size of the fragments of interest decreases to below 1000 base pairs (bp), however, the resolution deteriorates signifi ...

Production of pure plasmid DNA is a prerequisite for most laboratories engaged in recombinant DNA research. There are a number of procedures available for plasmid purification, all of which have common features; namely, (1) growth of plasmid-containing bacteria, (2) lysis of bacteria, (3) l ...

Elutips are essentially minicolumns of reverse-phase resin that offer an extremely rapid method for DNA purification and may often replace conventional techniques for manipulation of DNA. The Elutip-d columns commercially available are manufactured by Schleicher and Schuell. ...

Chloroplasts in common with other plastids and mitochondria have their own nucleic material, in the form of multiple copies of a circular DNA molecule, normally of 120–160 kbp.

In wheat, growth is from a basal meristem: The cells of the young shoot form a developmental array from the tip (oldest) to the base (youngest) of the leaf. In cells from a specific part of the leaf (and so of a certain age), chloroplast DNA is found to be replicating. In seedlings up to 10 d old, chloroplast DNA replicati ...

The techniques of plant molecular biology have advanced rapidly in the last 5 yr, and, for a number of plant species, including some crops, it is now possible to bypass traditional plant breeding techniques and introduce specific genes directly. The earliest reports of the transformation of pl ...

The formation of crown galls on many dicotyledonous plants, as a result of Agrobacterium tumefaciens infection, has been well documented (1). The transfer of a small T-DNA region of the Ti plasmid to the nuclear genome of the plant host (2) and expression of the T-DNA growth hormone genes are responsib ...

It is now possible to regenerate plants from protoplasts of a wide range of species. As a result, genetic manipulation by protoplast fusion in vitro is now a realistic proposition. This area is exciting because protoplasts from different origins can be fused together to form new genome combinat ...

The most commonly used systems to translate mRNAs in vitro are the rabbit reticulocyte lysate and the wheat germ extract (1,2 and see Vol. 2 in this series). Both these systems have a number of advantages, including the ease and cheapness of preparation, the relative absence of RNAse, the high level of rei ...

Isolated plant nuclei can be used for fundamental studies on the transcription apparatus. Total RNA polymerase activity can be measured using plant nuclei, and by using different α-amanitin concentrations in the enzyme assay, the individual RNA polymerase I, II, and in activities can be mea ...

Use of the Agrobacterium tumefaciens Ti plasmids to introduce foreign genes into many plant species is now firmly established (1). Ti plasmids, which are of tern single copy molecules, typically of 180–220 kilobases (kb), are divided into three main groups: nopaline, octopine, and agropine ty ...

The isolation of plant nuclei is a useful first step in many experiments concerned with the mechanism and control of gene expression in plants. For example, isolated nuclei can be used for the isolation of nuclear components such as chromosomal proteins (1), for the study of the processing of primary ...

Current research into the structure and function of plant genes involves the application of many elaborate techniques for gene cloning and analysis. The isolation of pure, intact plant mRNA is required at many stages in this process, e.g., for generation and screening of cDNA clones, for charac ...

Specific fragments of single-stranded DNA may be covalently immobilized to solid supports for various reasons. The support can be used as an affinity column for purifying DNA from a test sample, to detect viral or bacterial DNAs, or for antenatal diagnosis of genetic diseases such as sickle cell a ...

It was found (1) that biotinylated analogs of dUTP, which contain a biotin molecule covalently bound to the C-5 position of the pyrimidine via an allylamine linker arm, can be used as substrates for DNA polymerase. Figure 1 shows the structure of biotin-11-dUTP, a commonly used substrate for biotiny ...

The process whereby part of the energy evolved in a chemical reaction is emitted as light is known as luminescence. When luminol is oxidized by hydrogen peroxide in the presence of horseradish peroxidase (HRP), light is evolved (1). This is shown in Fig. 1. Luciferin not only enhances the reaction, but pr ...

Many research workers and clinicians working in routine laboratories are now using the “tools” developed by molecular biologists over the past 10 years to produce DNA sequences that can be used as probes to detect specific genes. Thus it is possible to screen food for salmonella (1) or other organi ...

Nucleic acid hybridization, the formation of a duplex between two complementary nucleotide sequences, is being increasingly utilized in the research laboratory and for the routine diagnosis of disease. Biotin-labeled nucleic acid hybridization probes have advantages over ra ...