生物化学技术

The polymerase chain reaction (PCR) provides an in vitro method for rapid enzymatic amplification of fragments of DNA. Microchip-based PCR devices (with reaction volumes from picoliters to microliters) have been realized using various combinations of silicon, glass, and/or plast ...

An ultrasensitive sandwich DNA array using highly fluorescent and photostable dye-doped silica nanoparticles is described. Compared to traditional sandwich arrays in which fluorophores have been used to signal target DNA molecules, the developed nanoparticle probes provi ...

This chapter describes the fabrication and use microfluidic chambers for cell migration and neuroscience research. Both microfluidic chambers are made using soft lithography and replica molding. The main advantages of using soft lithography to create microfluidic chambers are ...

This chapter outlines the methods and procedures for making a microfluidic and microfabricated biofuel cell. Commercially available screen-printing carbon inks are employed as electrodes by micromolding them onto glass microchips. The carbon ink electrodes are modified with ...

Musculoskeletal tissue-engineering strategies have recently focused on the use of biomaterial scaffolds capable of guiding growth and organization of mesenchymal stem cells (MSCs), which are precursors for connective tissues. This chapter describes the methods for culturi ...

This chapter introduces a microfluidic method to study the contraction of a single cardiac muscle cell (cardiomyocyte). This method integrates single-cell selection, cell retention, dye loading, chemical stimulation, and fluorescence measurement for intracellular calci ...

This chapter describes the design and fabrication of a passively driven microfluidic sperm sorter using soft lithographic microfabrication techniques. This self-contained device can separate motile sperm from nonmotile sperm and other cellular debris. The sorting system is s ...

This chapter provides a detailed description of a network of channels that includes a chamber for trapping beads in a microfluidic device. Instructions are included for the packing and use of the bead bed for solid-phase extraction (SPE). The SPE procedure may be used, e.g., as a filter to clean up a dirty sa ...

Fabrication of micromachined magnetic particle separators is introduced with an emphasis on magnetic particle-based bioseparation in microfluidic systems. The most attractive aspect of the magnetic separation technique in biochemistry and biotechnology is ease of manip ...

We describe a basic, fast, and reliable technique to isolate and characterize ribonucleoprotein (RNP) using antibody to a constituent protein. The antibody serves to immunopurify RNP from total cells or nuclear and cytoplasmic cell fractions under conditions that promote RNP integr ...

RNA-binding proteins can organize messenger RNAs (mRNAs) into structurally and functionally related subsets, thus facilitating the coordinate production of gene classes necessary for complex cellular processes. Historically, in vitro methods primarily have been used to id ...

Proteins interacting with messenger RNAs (mRNAs) affect their nuclear processing, export, translation efficiency, stability, or cytoplasmic localization. Such RNA-binding proteins are often modular, containing RNA-binding domain(s) and other functional modules. To ana ...

The 3′ poly(A) tail of eukaryotic messenger RNAs (mRNAs) acts synergistically with the 5′ cap structure to enhance translation. This phenomenon has been explained by the simultaneous binding of poly(A)-binding protein (PABP) and a cap-binding protein (eIF4E) to eIF4G that results in the circ ...

In cells, the poly(A) tail stimulates translation from messenger RNAs bearing a cap structure or viral IRES elements. This 3′ end-mediated stimulation of translation is not reflected in commonly used commercial cell-free translation systems prepared from rabbit reticulocytes or wh ...

Removal of the 5′ cap from a messenger RNA (mRNA) is an integral part of all mRNA decay pathways and can be a highly regulated event. Assays designed to assess decapping in vitro need to effectively resolve four products of mRNA decay: 7meGpppG produced by 3′–5′ shortening of the transcript by the exosome, 7m ...

Three types of exonucleases contribute to the turnover of messenger RNAs in eukaryotic cells: (1) general 3′-to-5′ exonucleases, (2) poly(A)-specific 3′-to-5′ exonucleases, and (3) 5′-to-3′ exonucleases. All three of these activities can be detected in cytoplasmic extracts from a variety of e ...

Most approaches to studying messenger RNA (mRNA) decay in vivo lack sufficient sensitivity to identify decay intermediates. The identification of such intermediates using in vitro decay systems can provide suggestive evidence for endonuclease-mediated degradation in vivo; to ...

RNA interference (RNAi) is a useful tool for degrading targeted messenger RNAs (mRNAs) and thus “knocking down” the abundance of the encoded protein. We have been using RNAi in cultured Drosophila cells to evaluate the effect of “knocking down” numerous mRNA processing factors on the alterna ...

Although RNA silencing was first discovered in plants, thus far it has been studied biochemically only in animals, where it is known as RNA interference (RNAi). In animals, two components of the RNAi pathway have been identified: Dicer, a multidomain RNase III that converts long double-stranded ...

The chromatin immunoprecipitation (ChIP) technique has been used to determine where and under what conditions DNA binding proteins associate with specific DNA sequences. Proteins are crosslinked in vivo with formaldehyde, and chromatin is then isolated and sheared. The protein of i ...