生物化学技术

Checkerboard DNA-DNA hybridization (CKB) is a technique that provides a simultaneous quantitative analysis of 40 microbial species against up to 28 mixed microbiota samples on a single membrane; using digoxigenin (DIG)-labeled, whole-genome DNA probes. Developed initially to st ...

Comparative mapping has been the primary approach for analyzing genomes of divergent species because gene linkage is often conserved over evolutionary time, a phenomenon known as synteny. Comparisons are based on computational detection of orthologous sequences between speci ...

In-gel hybridization with digoxigenin (DIG)-labeled probes has been shown to detect complementary DNA sequences in dried agarose gels. Gels dried at room temperature or at 55�C in an oven do not show detectable changes in the sensitivity of detection. However, gels dried under vacuum seem to lo ...

In situ hybridization is one of the most direct and reliable ways to ascertain the origin of the gene from complex mixed cellular systems. This method is essential for studying communities of uncultured microorganism in their natural ecosystem. In this chapter, we introduce our protocols for ...

In situ hybridization to messenger RNA (mRNA) in complex tissues, such as the brain, allows the localization of gene expression to functionally distinct regions. It has been difficult to measure relative changes in gene expression within these regions because of the poor cellular resolut ...

Many developmental and physiological analyses, population studies, and diagnostic tests can be performed by simply determining the presence or absence of a limited number of gene products. Here, we describe a rapid and sensitive procedure, based on the reverse transcription of total RNA ...

This chapter presents a protocol for the phylogenetic identification of microorganisms in environmental samples (water and sediments) by means of fluorescence in situ hybridization (FISH) with ribosomal RNA-targeted oligonucleotide probes and signal amplification (ca ...

The RNA electrophoretic mobility shift assay is a simple and rapid method for visualizing the existence of specific RNA-protein interaction. We have developed a useful method for the detection of mRNA-binding proteins using fluorescence-labeled synthetic RNA. In this method, RNA was p ...

Kinetic or real-time PCR continues to develop at a rapid rate since its development in the early 1990s. New applications are continually being found for this technique and it is replacing conventional PCR in many fields because of its speed, reduced hands-on time, and because the closed-tube form ...

Changes in copy number of genes contribute to the pathogenesis of various genetic disorders and cancer. The status of a gene has not only diagnostic value but sometimes directs treatment stratification. Although, for many years, Southern blot and fluorescence in situ hybridization were t ...

RNA interference (RNAi) is a natural mechanism, that is triggered by the introduction of double-stranded RNA into a cell. The long double-stranded RNA is then processed into short interfering RNA (siRNA) that mediates sequence-specific degradation of homologous transcripts. This ph ...

In routine molecular diagnostics, real-time PCR has made a major impact because of the faster time to the result; decreased hands-on time; and virtual elimination of the major issue in the early days of PCR, sample contamination from previously amplified DNA. The shorter time to the result for a real- ...

Gene expression microarrays are being used widely to address a myriad of complex biological questions. To gather meaningful expression data, it is crucial to have a firm understanding of the steps involved in the application of microarrays. The available microarray platforms are discu ...

There are more than a dozen formats available for the fluorescent detection of amplified DNA in kinetic (real-time) PCR. These chemistries are adaptable to most real-time PCR instruments and may offer benefits over the usual manufacturer-recommended chemistries for the instrument. T ...

DNA microarrays are well suited as a tool for analyzing functional gene diversity as well as community composition in aquatic environments. Microarrays allow for the semiquantitative characterization of target genes by means of specific hybridization of labeled target gene sequ ...

In the early 1990s, attempts to manipulate gene expression by researchers working in three different fields resulted in unanticipated gene silencing. Rather than ignoring such results, these researchers went on to document and further investigate the nature of such silencing, which w ...

The nematode Caenorhabditis elegans is often employed in investigations of diverse aspects of biology, including behavior, development, basic cellular processes, and disease states. The ability to utilize double-stranded RNA (dsRNA) to inhibit specific gene function in this or ...

The discovery of RNA interference (RNAi) has greatly simplified the process of suppressing genes in many experimental systems, including Caenorhabditis elegans, Drosophila, and mammalian cells. A sequence-specific nuclease complex, called the RNA-induced silencing compl ...

Trypanosoma brucei, a flagellate protozoa of the family Trypanosomatidae, has become one of the model systems for unicellular pathogens to study fundamentally important biological phenomena. Currently, the method of choice to examine gene function in these organisms is RNA interf ...

RNA interference (RNAi) is now a popular method for silencing gene expression in a variety of systems. RNAi methods use double-stranded RNAs (dsRNAs) to target complementary RNAs for destruction. In mammalian systems, very short dsRNAs (22–25 bp) such as short interfering RNAs (siRNAs) or sh ...