生物化学技术

Hybrid arrested translation (1) is a means of identifying recombinant DNA clones by their ability to hybridize to, and thus prevent the translation of, a specific messenger RNA in a cell-free system. In this respect the approach is similar to “hybrid selection,” also known as “hybrid release” (see C ...

The characterization of specific DNA sequences by means of hybridization techniques is of great relevance in the development of molecular biology. Because of its simplicity, the method described by Southern in 1975 (1) is widely used for such purposes. This method, also known as the Southern b ...

Nucleic acid hybridization is a very potent technique that can be used for the identification of DNA and RNA species with varying degree of homology and for the estimation of relative amounts of nucleic acid with known homolgy. In most cases, single-stranded (ss) (denatured) DNA or RNA is bound to a fil ...

Many laboratories are currently interested in cloning cellular genes that code for polypeptides that may have academic or therapeutic use. Once the intact gene is isolated, usually in the form of a cDNA clone, it has to be engineered into an expression cassette that is capable of functioning when i ...

Many techniques have been developed to transfect mammalian cells with DNA (1–2). The most commonly used method is to expose cells to a coprecipitate of DNA and calcium phosphate (3). This technique works very well with both genomic and recombinant DNA sequences for some cell lines, e.g., mouse L-cell ...

A major use of bacterial expression vectors is the expression of fragments of DNA against which monoclonal or polyclonal antibodies may be raised. In some cases this is the only way of generating an antibody, e.g., when investigating an open reading frame of DNA for which the gene product is unknown. By ra ...

Monoclonal and polyclonal antibodies are important tools in molecular and cell biology, allowing protein molecules to be purified (1), identified in immunoblots of gels (2) and sections of biological material (3), and probed for functionally active regions. Studies on the nature of anti ...

Most methods for screening libraries of cDNA clones rely on the selective binding of either nucleic acid probes to cDNA molecules or antibodies to the polypeptide gene product encoded by the cDNA. Although screening of cDNA libraries with synthetic oligonucleotide probes is undoubted ...

Making libraries of DNA fragments in various cloning vehicles is a basic experimental procedure in molecular biology, yet the methods involved often yield disappointing results, especially for the beginner. When screening for rare DNA molecules, large libraries must be constructe ...

With the development of improved techniques for the construction of cDNA libraries representing rare messenger species has come increased demand for screening techniques to isolate specific cDNAs. A variety of techniques has been developed:

Although the avidin-biotin detection system has been used for immunochemical staining of tissues for some time, its use for amplification in immunoassays was not described until 1979 (1). The biological basis for the system is the very tight binding of four molecules of biotin to one of avidin. Th ...

To obtain a cDNA clone of an mRNA, the mRNA must be copied faithfully into DNA, and the cDNA library must be large enough to represent the abundance class that contains the mRNA of interest. For example, in tobacco, Goldberg (1) has shown that it is possible to divide the mRNA population into three classes with mo ...

Many situations arise in recombinant DNA research in which it is necessary to identify the mRNA encoded by a particular cloned DNA sequence. Cloned DNA fragments can be characterized by hybridization to mRNA, the complementary mRNA then being identified by translation in vitro. There are two ...

To study the organization and regulation of eucaryotic genes from cloned DNA, vectors capable of maintaining large inserts have been constructed, largely from bacteriophage λ, where nonessential intergenic regions were identified. It had been noted that only λ DNA 37–52 kb in length (or 78– ...

The ideal genomic library should consist of a series of clones containing overlapping sequences that are representative of the entire genome. Such an ideal state can be approached by cutting the DNA randomly and cloning large pieces of this DNA into a suitable vector (1). The DNA can be cleaved either ...

The development of the chemistry for solid-phase oligonucleotide synthesis has enabled its widespread use in molecular biology laboratories for construction of novel linkers, site-directed mutagenesis, and many other techniques. The material obtained from a particular syn ...

Recent advances in molecular biology have made it possible to construct complete gene libraries for any organism that uses DNA as its carrier of genetic information. A gene library should contain a large number of cloned DNA fragments that in total contain the entire donor genome. The construct ...

Oligodeoxyribonucleotides may be synthesized on a solid support, which allows for the elongation of the chain without intermediate purifications. The exocyclic amino groups on cytosine and the purines are protected as the alkali-labile benzoyl or iso-butyryl amides. The 3′-OH group ...

During the past few years, synthetic DNA, used as a primer in DNA polymerization, in site-directed mutagenesis or as a probe in gene selection, has assumed a central role in recombinant DNA technology (1). This was made possible by the development of methods for efficient solid phase chemical synth ...

The initiation of transcription of eukaryotic genes by RNA polymerases is controlled by complex interactions between nonhistone proteins and specific regulatory DNA sequences (promoters and enhancers) (1). In order to characterize and purify such transacting protein factors, a ...