生物化学技术

The contamination of food with organisms, such as Salmonella, Listeria, and Escherichia coli, continues to be an area of concern for consumers and food producers. Historically, the identification of such organisms has been done using conventional culture methods and biochemical tech ...

This chapter describes a nonradioactive method for the localization of mRNA in whole mouse embryos. It employs riboprobes labeled with digoxigenin, a steroid-like moiety not found in animal tissue. Digoxigenin-containing probe is visualized with a conjugate of antidigoxigenin F ...

Nonradioactively labeled probes offer several advantages compared to radioactive ones, as they show long stability, high morphological resolution, and rapid developing time. There are different types of nonradioactive labeling methods available, although recently digo ...

Digoxigenin is widely used as a nonradioactive label for in situ hybridization to locate DNA sequences along chromosomes in plants (e.g., 1–3) and animals. In many cases, its use is similar to biotin (chapter 25, or e.g., 4), but digoxigenin labels may give lower unspecific background signal and there ...

Chromosomal in situ hybridization enables determination of the presence and location of DNA sequences complementary to a labeled probe along chromosomes and within interphase nuclei. The use of directly fluorochrome-labeled probes means that sites of probe hybridization can be d ...

In situ hybridization of biotin-labeled probes to plant chromosomes is a powerful technique enabling the physical mapping of DNA sequences and the “tagging” of chromosomes for identification (1). The method described here is based on Rayburn and Gill (2) and is particularly successful for ...

An important factor contributing to the success of DNA:DNA in situ hybridization is the preparation of clean chromosome spreads (1,2). The chromosomes and nuclei should be well separated and free of cytoplasm, debris, and dirt (Fig. 1). Nonspecific signal is deposited on cytoplasm and cell deb ...

Success with hybridization of DNA sequences in situ to plant chromosomes depends on many factors, but of foremost importance is the quality of the chromosome preparation. If this is not of high standard in terms of the number of metaphases achieved and the cleanliness of the background, there is re ...

Oligonucleotide synthesizers are now commonplace with the result that oligonucleotides can be obtained readily, obviating the need for complex molecular biological techniques. However, the choice between long probes or oligomers depends, to a large extent, on the application; ol ...

DNA probes can be labeled using the modified nucleotide, fluo-rescein-11-dUTP, and subsequently detected with enhanced chemiluminescence (1) using an antifluorescein antibody conjugated to horseradish peroxidase (2). The use of enhanced chemiluminescence avoids the fadi ...

The use of probes directly labeled with horseradish peroxidase (HRP) in conjunction with enhanced chemiluminescence (ECL) allows a flexible approach to hybridizations and detections. This is shown by the diversity of applications in which this labeling and detection system has been ...

The probing of RNA gel blots (also called Northern blots) with labeled nucleic acids provides data on the relative levels of steady state gene expression, and on RNA processing. The principle of the technique was first described by Alwine et al. (1). The protocol described in this chapter demonstra ...

The following procedure describes the hybridization of digoxigenin-labeled RNA minisatellite probes (see chapter 12) to Southern blots of different species and visualization of the DNA banding patterns by color detection. The method can be applied to both multi- and single-locus pr ...

Probes prepared with either digoxigenin- or biotin-modified nucleotides can be hybridized to Southern blots to detect target nucleic acid sequences. These methods offer an attractive alternative to “radioactively tagged“ probes in terms of safety, cost, and efficiency. Most prev ...

Oligonucleotides are a convenient alternative to conventional, cloned DNA probes since they can be rapidly synthesized in large quantities relatively cheaply. They can be made to very specific regions of the genome, without reference to restriction enzyme sites, and are therefore of pa ...

It is the aim of this book to provide working, reliable, protocols for nucleic acid analysis that avoid the use of radioisotopes. It is hoped that in doing so, the widespread use of this technology will be encouraged in areas where the older procedures, based on radioisotopes, are not practicable.

The analysis of RNA is central to a wide range of molecular-biology studies, because It 1s often important to obtain informatlon about the expression of genes in hving organlsms Filter hybrldlzatlon of size-separated RNAs with a labeled nucleic-acid probe, a techinque known as northern-bl ...

The detection and identification of specific nucleic acid sequences is routine in molecular biology research The principles that govern molecular annealing (or molecular hybridization) are used in many apphcatlons to the study of nucleic acids. Under sultable conditions, two sin ...

Many important advances in molecular biology would not have been possible wlthout the use of radioisotopes It is relatively simple to substitute a radtoacttve isotope into a nucleotide to produce a molecule that has the same biological properties as the unlabeled molecule (see Chapter 6) T ...

The use of nucleic acid or gene probes as cloning and diagnostic tools has proven to be a powerful technique for lsolatlon and mampulatlon of genes, as well as for rapid detection and ldentlficatlon of bacteria and other mlcroorgamsms, with wide apphcatlons in molecular biology, medical, and env ...