遗传学实验技术

The polymerase chain reaction (PCR) is fast becoming a standard protocol in many laboratories for cloning and analysis of small quantities of DNA (1). It involves the exponential synthesis of linear DNA molecules from double-stranded DNA templates using specific oligonucleotide pri ...

The cloning of eukaryotic DNA in bacteria or by the polymerase chain reaction (PCR) inevitably results in the loss of much information, Protein-DNA contacts and cytosine methylation patterns, both important in gene regulation and other cellular processes, are not reproduced in these cl ...

The expectation that genome sequence analysis will provide an increasingly useful approach to the characterization of biological material has led to suggestions that the complete genome sequences of organisms ranging from Escherichia coli to Homo sapiens should be determined. W ...

Bacteriophage lambda is widely used as a vector for both genomic and cDNA cloning. However, although lambda is highly efficient for cloning, its use for direct sequencing is limited because of a lack of reliable sequencing methods. Although various methods for sequencing directly in lambda h ...

The underlying principle of both main DNA sequencing methods by Sanger (1) and Maxam and Gilbert (2), is the ability to fractionate and resolve long, single-stranded DNAmolecules that differ in length by only one nucleotide. Denaturing polyacrylamide gels have been reported to give interp ...

The products of sequencing reactions are separated on thin, low percentage (usually 6%) polyacrylamide gels. Normally, these gels are 40–50 cm in length, 20 cm wide, and between 0.3 and 0.4 mm thick. Longer gels (to enable more sequence to be read from a single run) up to 100 cm in length can be poured, as can wider gels ( ...

Tuq DNA polymerase is a highly stable polymerase isolated from the thermophilic organism Thermus aquaticus. Because of its unique properties this enzyme has been extensively used for amplification of DNA fragments by the polymerase chain reaction (PCR). In addition, the thermostabi ...

The dideoxy chain-termination method of DNA sequence analysis involves the synthesis of a DNA strand by enzymatic extension from a specific primer using a DNA polymerase (1). Several different enzymes are available for this purpose each having different qualities and properties. The use ...

Why produce transgenic fish? There are two chief reasons for introducing novel genes into animals. The first is as a means of increasing knowledge of gene regulation in that particular group of organisms. The second is that transgenic induction may involve some economic benefit from the modif ...

Although transgenesis studies of fish started relatively later compared to those of mammals, they have not been completely neglected, and in recent years, many publications have appeared in this field. Reviews of transgenesis in fish are given by Maclean et al. (1), Hew (2), Maclean and Penman (3), a ...

In contrast to many organisms, Drosophila embryos do not integrate injected DNA at an appreciable frequency. For this reason, the generation of germ-line transformants has relied on the utilization of transposable elements to effect the chromosomal integration of injected DNA (1,2). T ...

Random primed labeling of DNA has now almost superseded the method of nick translation of DNA. Random primed labeling, based on the method of Feinberg and Vogelstein (1) is a method of incorporating radioactive nucleotides along the length of a fragment of DNA. Random primed labeling can give spec ...

In recent years there has been a large increase in the number of nonradioactive methods of detection of nucleic acids (see Chapter 45). Despite this, the vast majority of laboratories still utilize radioactive labeling. Although it is possible to foresee that in 10–15 years radioactive nucle ...

Histological examination is important to detect and differentiate among different types of tissue changes at the microscopic level. An enlarged organ can be hyperplastic, hypertrophied, or neoplastic. It can also be merely inflamed or infected. Only a histological examination can d ...

Postmortem examination of transgenic animals should ideally be performed by or with the help of a trained pathologist. This is because although the steps in the examination can be manually performed by anyone, the interpretation of the findings can only be done with the knowledge of veterinary ...

The use of S1 nuclease to map the start site of a transcription unit is a well-established technique. Based on the method of Berk and Sharp (1), it has undergone many refinements over the years. S1 nuclease mapping requires a relatively detailed knowledge of the gene structure and sequence data (or a very g ...

The methods described in Chapters 39, 40, 43, and 44 can only provide information about steady-state levels of transgene RNA. Any differences in specific RNA levels observed in different tissues of a transgenic organism, or any changes in RNA level as a consequence of a physiological or developme ...

Methods used to study gene expression rely on two basic conditions. First, sufficient tissue must be available for analysis, and second, the gene must be expressed at a level high enough to be detectable by the method used. Where the gene of interest is expressed in small tissues or groups of cells, for exam ...

Using slot blots, RNA can be applied, unfractionated, to a solid matrix. Slot blotting can be used as a rapid method for analyzing changes in transgene RNA quantity following developmental or physiological changes. However, it can only be applied to animals that bear a transgene that has hybridiz ...

The analysis of RNA is often the easiest and quickest method of determining the pattern of transgene expression in a transformed organism. Such analyses demand that the RNA extraction method be relatively rapid and painless. Some RNA preparation methods, for example, those that require a CsC ...