生物化学技术

Oligonucleotide analogs bearing modified phosphodiester linkages have been the focus of considerable interest in the antisense field, with methylphosphonates and phosphorothioates being the most extensively studied derivatives to date (1–3). These modifications, whe ...

Over the last 30 years or so, the chemical synthesis of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) has constituted one of the most challenging problems in the field of the synthetic organic chemistry of natural products. In more recent times, attention has been focused not on “natural” ...

The value of oligonucleotides as therapeutic agents has become increasingly apparent over the past decade (1–3). In order to be useful as therapeutic agents, oligonucleotides must possess a number of properties. These include: 1. Resistance

There are three constitutionally isomeric forms of internucleoside phosphorothioate linkages. Replacement of either the 3′- or 5′-oxygen in a phosphodiester linkage, 1, results in the respective thiolo structures 2 or 3, whereas substitution of a nonbridging oxygen with sulfur gives a ...

Julian Barnes wrote A History of the World in 10 1/2 Chapters (1). Oligonucleotides on this score warrant at most a comma, a literary augenblick, but lacking the skill of the aforementioned author and still using a broad brush, perhaps ten pages or so may suffice to paint the picture. This is an essay or perhaps c ...

The selection of an appropriate labeling reagent for a particular experiment, for the most part, depends on the sensitivity and resolution required. For maximum sensitivity in filter hybridizations radioactive labels, such as phosphorous 32, are still the most widely used.

This chapter describes a DNA extraction method that can be used both on freeze-dried leaves and on fresh leaves, and is based on the method of Saghai-Maroof (1), modified by David Hoisington and Jack Gardiner at the University of Missouri at Columbia (personal communication). The scale of the extra ...

The direct labeling of nucleic acid probes with horseradish peroxidase (HRP) was first described by Renz at EMBL in 1984 (1). The methodology was combined with enhanced chemiluminescence (2) (a light producing HRP catalyzed reaction; see chapter 20) allowing the detection of specific hybr ...

Probes labeled with biotin have been used for hybridizations to Southern blots and to chromosomes in situ since the early 1980s (1). Nowadays, the labeling of double-stranded DNA probes with biotin is made easy by the availability of labeling kits that provide everything required for nick tran ...

The technique of DNA fingerprinting detects a large number of independent hypervariable minisatellite loci throughout the genome in a single hybridization experiment and is an extremely powerful and sensitive means for genetic analysis of very many animal and plant species (a broad o ...

Oligolabeling using random hexanucleotide primers (1) is an effective method for producing DNA hybridization probes of high specific activity, although most previous variations have utilized radionuclides as the mechanism for modifying the incorporated nucleotides. With ...

The polymerase chain reaction (PCR) (1) is an efficient method for copying a fragment of DNA when primers exist that flank the target sequence. We have found that since most cloning vectors utilize the lacZ gene with a synthetic multiple cloning site (2), a single pair of primers will amplify sequences ...

Most molecular probes are produced by cloning, or subcloning, the required “foreign” DNA fragment (i.e., the DNA sequence that will be used as the final probe) into a bacterial plasmid. This chapter describes the basic procedures necessary to produce plasmids of sufficient purity to label dire ...

RNA gel blots (often referred to as Northern gel blots) are frequently used in the analysis of gene expression, for example when investigating specificity of gene expression, quantification of transcription, and analysis of RNA processing. Electrophoresis of RNA through agarose gels ...

Most RNA in a mammalian cell consists of 28S, 18S, and 5S ribosomal RNAs together with tRNAs and other small ubiquitous RNAs. The remainder (

Efficient extraction of high quality RNA from a variety of plant tissues is an important first step in many procedures, such as analysis of gene expression, cDNA library construction, and in vitro translation. This procedure, which is essentially as described in Draper et al. (1), involves grind ...

In 1975, Edwin Southern published a paper that revolutionized the molecular analysis of the genomes of organisms (1). This procedure enabled the detection of which fragment of DNA (out of the millions that compose the genome of a higher organism) contained sequences related to that of a radioact ...

The ligase chain reaction (LCR) is a novel DNA amplification system developed jointly by Abbott Laboratories (North Chicago, IL) and Biotechnica International, Inc. (Kansas City, MO) (1). By amplification with specially haptenated detection probes, LCR can detect less than 100 target DNA ...

The analysis of DNA-based polymorphisms is integral to the construction of molecular genetic maps. Currently, the method of choice is the restriction fragment length polymorphism (RFLP) assay. The RFLP method only allows the detection of DNA sequence polymorphisms by Southern blot hy ...

The introduction of polymerase chain reaction (PCR) technologies (1–4)has greatly simplified the analysis of gene sequences. Prior to PCR, comparison of genes and their alleles required lengthy, tedious, and somewhat difficult procedures. These steps included DNA or RNA purificat ...