phosphorus-radioisotopes and deoxyuridine-triphosphate

One of the defining biochemical characteristics of apoptosis is the degradation of chromatin into regularly sized (oligonucleosomal and approximately 30- to 50-kb) fragments. Because destruction of the genome represents a clear commitment to death, considerable interest has focused on this component of apoptosis and numerous assays have been developed to assess the relevant nucleases involved. These assays fall into two major categories: (1) those independent of chromatin structure and (2) those dependent on chromatin structure. The chromatin-independent assays (plasmid degradation assay and radioactive gel assay) examine the ability to degrade naked DNA and are advantageous because of their simplicity and speed and ability to analyze single nucleases or mixtures of nucleases. However, these assays do not mimic the conditions present in normal cells and consequently do not assess the ability of an enzyme to function in apoptosis. In contrast, chromatin structure-dependent assays (nuclear autodigestion and HeLa nuclei assay) present intact chromatin to either endogenous or exogenous enzymes and assess the ability to degrade chromatin in a manner that recapitulates the genomic destruction seen in vivo. Detailed protocols are discussed for both classes of assays. These assays have been instrumental in the identification of several apoptotic nucleases.

Topics: Animals; Apoptosis; Cell Line; Cell Nucleus; Chromatin; Deoxyribonucleases; Deoxyuracil Nucleotides; Dexamethasone; DNA; DNA Fragmentation; Electrophoresis, Agar Gel; HeLa Cells; Humans; Indicators and Reagents; Phosphorus Radioisotopes; Plasmids; T-Lymphocytes

A method for fluorescent labeling of PCR products has been developed. This method consists in a two-step procedure in which a first exponential classical PCR is followed by a "linear amplification". This second step relies on incorporation of fluorescent dNTP (dUTP or dCTP) in order to label the product on only one strand. The products can be applied without prior purification directly to a gel on a fluorescence-based automated DNA sequencer, for length and allele determination. The reliability of the results equals those of the classical 32P or fluorescent primer labeling methods, and the method is definitely less costly. Since the interpretation of the results is easier than with the method consisting in a fluorescent dNTP uptake in both strands in a single PCR, the present strategy should prove useful in mapping projects requiring analysis of a large number of microsatellites.

Topics: Animals; Base Sequence; Biotechnology; Deoxycytosine Nucleotides; Deoxyuracil Nucleotides; DNA Primers; Dogs; Fluorescent Dyes; Microsatellite Repeats; Phosphorus Radioisotopes; Polymerase Chain Reaction; Polymorphism, Genetic