遗传学实验技术

Using transgenic animals as the source of recombinant proteins has several specific advantages. Large amounts of proteins can be obtained, essentially from milk. These proteins are often properly processed. They are in a number of cases correctly folded, assembled, cleaved, glycosyl ...

The ability to obtain essentially unrestricted quantities of a cloned protein via expression in a bacterial, fungal, or eukaryotic cell expression system facilitates the structural and functional characterization of that protein. The rapidly increasing availability of dive ...

There are many methods for creating animal models of human genetic disease, and it is not possible for a short chapter to provide complete details of the methods used. There are four general approaches that will be discussed. One involves embryonic stem cells and another transgenic mice, each of whi ...

Drosha is a member of the ribonuclease (RNase) III family. Like other RNase III proteins, Drosha is a double-stranded RNA (dsRNA)-specific endonuclease that introduces staggered cuts on each strand of the RNA helix (1,2). RNase III proteins are classified based on domain organization. Drosha ...

Current methodologies in first-generation adenoviral gene transfer, however novel their approach to vector delivery, are ultimately limited by the purity of the vector being delivered. Purity in this case is defined both from the standpoint of genetic homogeneity, and from the absence ...

Methods detailed in this chapter relate to the use of Lariat-dependent Nested (LaNe) PCR to characterize unknown RNA or DNA sequence flanking known regions. A multitude of approaches designed to determine flanking sequences have been described in the literature. Variously, problems r ...

Effective gene delivery to mammalian cells is a necessary step in the development of gene medicines and therapy. A variety of techniques have been developed to enhance the transfection of exogenous DNA into mammalian cells. These techniques include viral, nonviral, and energy-based sys ...

Throughout the past six or seven years, our ability to study and understand the process of eukaryotic gene expression has been greatly enhanced by the use of reporter genes. In essence, a reporter gene encodes a protein product for which a sensitive and convenient assay is available. Transcripti ...

The gel retardation or electrophoretic mobility shift assay (EMS A) is a sensitive technique for studying protein–DNA interactions. Originally devised by Fried and Crothers (1) to study the kinetics of protein‐DNA interactions, the method relies on the stability of protein DNA complex ...

This chapter describes one method for high resolution photofootprinting of proteins or small drugs bound to DNA (1). DNA footprinting is an electrophoretic method that permits the visualization (footprint) of the binding site of a molecule site-specifically bound to DNA. The method ent ...

Genomic footprinting is an extension of the genomic sequencing technique and is used to study protein/DNA interactions in vivo 1) (Fig. 1) It gives information concerning the sequence of protein binding elements and the contact points between a protein and its DNA target extend. Proteins bou ...

The polymerase chain reaction (PCR) is a powerful method for amplification of DNA segments between two regions of known sequences (1, 2). In standard PCR, sequences bound by two unique oligonucleotide primers can be exponentially amplified. However, it is a prerequisite to know the DNA sequen ...

The SI nuclease is an endonuclease isolated from Aspergillus oryzae that digests single‐ but not doublesdeshstranded nucleic acid. In addition, it digests partially mismatched double‐stranded molecules with such sensitivity that even a single base pair mismatch can be cut and hence d ...

A great deal of attention in recent years has been focused on the mechanisms governing transcription. Repression and/or activation of specific genes, or sets of genes, represents a key regulatory step in such diverse processes as cell growth, differentiation, and response to external stim ...

The quantification of mRNA in mammalian tissues or cell culture is an important part of analysis of gene expression and is required to fully understand many biological processes, such as cell replication, growth, and differentiation or the effects of hormonal, dietary, and genetical fact ...

The isolation and characterization of RNA polymerases from the Salmonella phage SP6 and the E. coli phages T7 and T3 has 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 form. ...

The clonal analysis of cell populations in neoplastic disorders and in solid tumors is an important tool in understanding the origin and progression of disease. The use of glucose-6-phosphate dehydrogenase (G6PD) isozymes in females (1) has proven to be extremely informative in these stu ...

The copy numbers of DNA sequences (templates) can be determined quantitatively by a combination of PCR and temperature gradient gel electrophoresis (TGGE; 1,2; Chapter 20) as illustrated in Fig. 1. Briefly, quantitation is performed by addition of a calibrated amount of DNA “standard” to the t ...

The potency of gel electrophoresis to analyze conformational transitions of nucleic acids has opened up a new field of applications. Helix coil transitions and, at a refined level, minor variations because of mutations and mismatches, may be studied experimentally by solvent -gradient ...

The diagnosis of disease by direct analysis is an increasingly important branch of laboratory medicine. The detection of DNA sequence anomalies in different at-risk individuals may be effected by indirect techniques (e.g., linkage analysis), or by more direct methods involving the det ...