遗传学实验技术

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 ( ...

The dideoxy chain-termination method of DNA sequence analysis involves the synthesis of a DNA strand from a single-stranded template (1). The enzymatic synthesis is initiated at the site where an oligonucleotide primer anneals to the template. Traditionally, sequencing was perform ...

Our ability to generate single-stranded DNA (ssDNA) templates has been invaluable for procedures involving DNA sequencing and site-directed mutagenesis. Conventionally, this is carried out by subcloning the DNA region under analysis into vectors that are based on the single-stra ...

M13 bacteriophage has a single-stranded DNA (ssDNA) genome, and has proven an extremely useful vector from which to derive single-stranded templates for sequencing and site-directed mutagenesis. During infection of its host cell, the phage DNA replicates as a double-stranded interm ...

The bacteriophage M13 has been developed as a cloning vector system to obtain single-stranded DNA templates that are used for the dideoxy chain termination method of sequencing DNA (1,2). General aspects of bacteriophage M13 as a cloning vector system are reviewed in ref. 3, and the preparation of ...

Random primed labeling of DNA has now almost superseded the method of nick translation of DNA.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 spe ...

Plasmid purification by alkaline lysis is one of the most generally used molecular biology techniques. Traditionally, the cleanest plasmid DNA has been made by banding the DNA on a cesium chloride gradient. This, however, is time-consuming and expensive, so alternative plasmid purific ...

The classic alternative to the alkaline lysis method for plasmid DNA preparation (1; see Chapters 31 and 33) is that of Holmes and Quigley (2), and is commonly known as the rapid boiling method. This method is based on exactly the same principles as the alkaline lysis method. The cells are partially lysed, a ...

The use of an electrical field to permeabilize cells reversibly (electroporation) has become a valuable technique for transference of DNA into both eukaryotic and prokaryotic cells. Many species of bacteria have been successfully electroporated (1) and many strains of E. coli are routi ...

Size selection of DNA fragments is frequently required before ligation or labeling for the preparation of probes. Many methods are available for purifying DNA fragments following electrophoresis in agarose gels, including the use of low-melting agarose, electrophoresis onto DEA ...

Subcloning procedures are used to transfer DNA fragments from one vector context (plasmid, cosmid, or phage) to another. They are commonly used to construct expression systems, and to transfer fragments into specialized vectors for the preparation of hybridization probes and single- ...

The preceding chapters describe the construction of genomic and cDNA λ libraries. In this chapter we describe two methods for screening libraries. The first method can be used on both genomic and cDNA libraries and screens by sequence homology. To do this, the library is plated and then “lifts” are ma ...

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 ...

The synthesis of complementary DNA from mRNA templates by the action of reverse transcriptase (RT’ase) is a fundamental technique in molecular cloning. The prime consideration is that a large amount of long cDNA copies should be made. The quality and length of the cDNA product is largely depende ...

In situ hybridization (ISH) is a widely used technique that has great power in many applications, including diagnosis of viral infections (1), chromosome analysis (2), and mRNA analysis (3). Traditionally, researchers have used radioactive labels to prepare probes for ISH (4). Such probes c ...

In a slot blot, RNA is applied, unfractionated, to a solid matrix, and therefore, slot blotting is a rapid and sensitive method for analyzing changes in RNA quantity following developmental or physiological changes. Its disadvantage is that it cannot be used when the RNA sample contains additio ...

In the analysis of gene expression, the steady-state level of RNA transcripts is one of the most convenient parameters used to monitor the gene activity in cell lines and tissues. A variety of methods, such as S1 hybridization, RNase protection, and Northern blotting, can be used to measure RNA levels, ...

The chromosome banding patterns produced by certain DNA binding fluorescent dyes (primary stain) can be enhanced or modified by an appropriately chosen DNA binding counterstain (Fig. 1). The latter ligand is usually nonfluorescent or may be fluorescent, but not in the wavelength range of t ...

General characteristics of chromosome banding and banding techniques have been described in the previous chapter (Chapter 6). In general, fluorescence banding provides much the same information as banding using absorption staining, but fluorescence staining methods are gene ...

Chromosome banding is the use of special staining procedures to induce patterns of longitudinal differentiation along chromosomes, in the absence of any structural differentiation (1,2). Specific chromosomes have characteristic patterns that permit their identification ...