生物化学技术

The enzyme-linked immunosorbent assay (ELISA) is typically used to detect and quantify antigen within biological fluids. Among the ELISA’s attributes are a very high level of sensitivity and robustness as well as being an extremely cost-effective assay that can be performed with only bas ...

In situ hybridization is a method used to localize nucleic acid sequences in cells or tissue sections. The first in situ hybridization experiments were described by two groups of researchers in 1969, John et al. and Gall and Pardue (reviewed in ref. 1). Radioactively labeled RNA probes were used to loc ...

The rapid development of efficient, automated DNA-sequencing methods has strongly advanced the genome-sequencing era, culminating in the determination of the entire human genome in 2001 (1,2). An enormous amount of DNA sequence data are available and databases still grow exponenti ...

By virtue of its high-resolving capability, short analysis time, and advanced instrumentation high-performance liquid chromatography (HPLC) has become a versatile technique for the separation and characterization of nucleic acids. Among the various chromatographic mode ...

The molecular diagnosis of hereditary and somatic disorders is a rapidly expanding field in modern medicine. Mutations in more than 1500 genes have been found to be associated with human diseases, and this figure is likely to rise significantly over the next few years as the genetic basis of more con ...

The invention of real-time polymerase chair reaction (PCR) has revolutionized the quantification of gene expression and DNA copy number measurements. However, after the first documentation of real-time PCR in 1993 (1), it took several years for this method to become a mainstream tool. PCR ge ...

The genomics revolution is transforming epidemiology, medicine, and drug discovery (1–7) and there is an ongoing refocusing of effort away from family-based linkage studies toward population-based genetic association studies for complex phenotypes (3,8–11). The generation of n ...

Many techniques are available to efficiently pick up (point) mutations, all with their own strong and weak points and limitations. The most powerful qualitative techniques include denaturing gradient gel electrophoresis (DGGE) (see Chapter 8), chemical cleavage of mismatches (CC ...

Direct nucleotide sequencing is the reference standard critical to all molecular biology whether it is used for the elucidation of an entire genome or the characterization of a specific mutation. Sequencing protocols were initially developed using either dideoxy nucleotides (1) or c ...

Protein blotting, the transfer of proteins from a separating gel onto a thin uniform support matrix, first appeared in 1979. Continuing the geographic theme following Southern's publication of his method for the identification of specific DNA fragments (1) in 1975 and the introduction of N ...

This chapter describes the separation mechanisms used for DNA electrophoresis. The focus is on the concepts that may help the researcher understand the methodology, read the theoretical literature, analyze experimental data, identify the relevant separation regimes, and/or des ...

The analysis of polymerase chain reaction (PCR) products by capillary electrophoresis (CE) is often compromised by the presence of a high concentration of salt. Typical PCR reactions contain 50 mM of KCl and 1.5 mM of MgCl2, which cause irreproducible electrokinetic injections and migrat ...

The fundamental role of any separation process is the isolation and quantitation of the individual components of a mixture. In the analysis of DNA by capillary electro-phoresis (CE), the migration time and the sample quantity are the basic parameters obtained from any electropherogram. M ...

Microchip-based capillary electrophoresis (CE) systems have enjoyed impressive advancement in the past few years (1–3). Up until now, CE array chips have been fabricated based upon the well-understood behavior of a single channel chip system. Different materials (e.g., silicon , glass , a ...

On May 9,1998, PE Biosystems announced that they would introduce a new instrument based on “breakthrough DNA analysis technology” (1). Over 700 orders for the new sequencer were received in the first 5 mo of production, which make this instrument one of the most successful new products in the history of ...

In this chapter, we examine important parameters that affect the performance of replaceable polymers currently used for DNA sequencing by capillary electrophoresis (DNASCE). Background on physical models used to describe polymer solutions and DNA migration in semi-dilute poly ...

Over the past decade, fluorescent end-labeling of DNA fragments has evolved into the preferred method of DNA detection for a wide variety of applications, including DNA sequencing and polymerase chain reaction (PCR) fragment analysis. One of the advantages inherent in fluorescent det ...

The charge density of nucleic acids is uniform and constant for nucleic acid molecules of varying length. Therefore, they typically migrate at the same velocity in response to an electrical potential in free solution. Capillary electrophoretic (CE) separation of nucleic acid species re ...

In traditional slab-gel electrophoresis, the gel has two functions: it serves as an anticonvective medium, as well as a sieving matrix that provides separation. Because of the low convection, CE permits a gel-free separation at high voltages, and thus yields unprecedented resolution and sp ...

Since its introduction in 1930 by Tiselius (1), slab-gel electrophoresis (SGE) has been utilized as an important separation technique in molecular biology for the resolution of DNA fragments, polymerase chain reaction (PCR) products, polynucleotides, proteins, and other compoun ...