遗传学实验技术

Patients suffering from Legionnaire’s disease initially experience nonspecific symptoms such as fever, myalgia, and headache. The predominant clinical finding is pneumonia with symptoms ranging from a mild cough and slight fever to diffuse pulmonary infiltrates and multiorg ...

The human hepatitis C virus (HCV) is an RNA virus discovered in 1989 that accounts for the majority of posttransfusion and sporadic non-A non-B hepatitis. More than half of patients with acute HCV infection develop a chronic course that is associated with a high risk of developing liver cirrhosis and ...

Genetic transformation of plants has enormously matured within the last few years. It began with the introduction of genes that imparted resistance to cultivated plants against herbicides and pathogenes. More recently, there are approaches to modify the quality of plants, such that use ...

The polymerase chain reaction (PCR) has proven to be an extremely powerful tool in the field of virus detection. It has been employed in a variety of situations where previous techniques were too slow, too cumbersome, or simply nonexistent. Some of these are as follows.

The identification of mutations is very important in such aspects of molecular biology as medical diagnostics, ascertaining structure/function relationships, population genetic studies, and in confirming the authenticity of new candidate genes. Presently there are a varie ...

Fingerprinting techniques are essential tools in the investigation of transmissible diseases. Unless one is able to track a pathogenic organism from its reservoir, through its vectors and carriers into infected hosts, it is impossible to define an organism’s epidemiology. In the abse ...

World-wide enteric pathogens are a leading cause of potentially preventable morbidity and mortality. They are responsible for an estimated 700-1000 million episodes of diarrhea and cause four to five million deaths each year (1). Infants and young children in the developing world are par ...

Since the discovery of the doublehelix structure of DNA (1), no single event has had the same impact on the field of molecular biology as the rediscovery by Kary Mullis in the early 1980s of the polymerase chain reaction (PCR) (2-4), which was first published in principle by Keld Kleppe in 1971 (5). This elegant ...

Silver stains are useful for the detection of nanogram amounts of proteins or nucleic acids in acrylamide gels or on various membranes. They have been shown to be more sensitive than organic stains in detecting proteins and DNA. They are capable of detecting as little as 0.03 ng/mm2 of DNA (1). In addition, ...

The isolation and characterization of RNA polymerases from the Salmonella phage SP6 and the E. coli phages T7 and T3 have revolutionized all aspects of the study of RNA metabolism (1–6). Indeed, it is now possible to generate unlimited quantities of virtually any RNA molecule in a chemically pure fo ...

In this reaction, the enzyme terminal transferase is used to introduce a short tail of fluorescein nucleotides to the 3′-end of an oligonucleotide (1) followed by horseradish peroxidase-catalyzed enhanced chemiluminescent detection of the labeled hybrids. This approach is used in the ...

A range of labeling and detection systems for nucleic acids, many of which show specific advantages for particular applications is currently available to the researcher. The labels generally fall into one of two categories: primary labels, in which a detectable signal, such as a radioisoto ...

The efficiency of incorporation of fluorescein-11-dUTP with both random prime labeling of long probes (Chapter 8) and 3′-tailing of oligonucleotide probes (Chapter 11) can be estimated using the following rapid labeling assay. The basic protocol shown is for use with random prime labeled ...

The 6xHis/Ni-NTA system is a fast and versatile tool for the affinity purification of recombinant proteins and antigenic peptides. It is based on the high-affinity binding of six consecutive histidine residues (the 6xHis tag) to immobilized nickel ions, giving a highly selective interac ...

Site-directed mutagenesis is a powerful technique by which specific changes can be generated in a target DNA molecule for either structure-function investigations or for creating and deleting endonuclease restriction sites. There have been several oligonucleotide-direct ...

There are two basic enzymatic activities that are used to end-label DNA with radioactive phosphate (32P). The enzyme T4-polynucleotide kinase will use the substrate ATP to add a phosphate group (the gamma-phosphate of the ATP molecule) preferentially to the 5′ ends of the molecule. The enzyme DNA ...

Efficient completion of large DNA sequencing projects has been greatly facilitated by the development of fluorescence-based dideoxynucleotide sequencing chemistries and instruments for real-time detection of fluorescence-labeled DNA fragments during gel electro ...

Since DNA sequencing has rapidly become standard practice in many laboratories, a large variety of new cloning vectors, sequencing strategies, and techniques have been developed to allow more efficient sequencing of a large variety of DNA templates. Despite a current focus on the automat ...

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 ...

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