Southern、Northern印记.doc

Southern and Northern Blotting DNA and RNA blotting, nucleic acid blotting. Figure 1 The southern and northern blotting techniques. Enzymatically digested DNA or denatured RNA is fractionated by gel electrophoresis. After transfer and fixation of the DNA fragments or RNA to a membrane support, the membrane is incubated with a labeled DNA or RNA probe that is specific for the target of interest. Because the probe hybridizes to only the target fragment/molecule, a specific band is visualized following detection. Figure 2 Capillary transfer of DNA or RNA from agarose gels. Buffer is drawn from a reservoir via a paper wick and passes through the gel into a blotting material (e.g. paper towels, blotting paper). The DNA or RNA is eluted from the gel by the flow of buffer and is deposited onto a membrane. A weight applied to the top of the blotting apparatus ensures a tight junction between the layers of material used in the transfer system. Short DescriptionNucleic acid detection can be performed using a technique known as blotting, which is the transfer of DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) onto a membrane support. When this procedure is used to examine DNA or RNA it is referred to as Southern or northern blotting, respectively. CharacteristicsSouthern blotting is the transfer of DNA from an agarose gel to a membrane support (1). Since its inception, Southern blotting has been used to determine gene copy number, detect restriction fragment length polymorphisms, determine modifications to DNA (e.g. methylation), and detect the presence of genetically inserted transgenes. It also is used to study RNA splicing and antibody and T cell receptor formation, and has aided in the identification of gene rearrangements associated with a variety of human genetic disorders and cancers. Northern blotting is the transfer of RNA from an agarose gel to a membrane similar to that used in Southern blotting (2). Northern blotting is used to evaluate gene expression in a manner that is both qualitative (in which tissues or cells, or under which physiological conditions a gene is expressed) and quantitative (level of gene expression). This method also is used to detect the expression of foreign genes in transgenic organisms and is a useful adjunct to complementary DNA (cDNA) cloning because mRNA molecular weights can be compared with those of cloned DNAs. The basic principles of Southern and northern blotting are similar; however, there are a few notable exceptions. RNA is more labile than DNA and is very sensitive to degradation by enzymes called RNases. RNA degradation reduces the quality of data and the ability to quantify message expression. RNA molecules often form secondary structures that significantly alter their mobility, requiring gel electrophoresis to be performed under denaturing conditions (in the presence of glyoxal or formaldehyde). With Southern blotting, genomic DNA is digested with restriction enzyme(s) to produce small fragments that can be easily fractionated, whereas RNA is analyzed as an intact molecule. Genomic DNA also does not require many of the special handling precautions necessary with RNA. The general schemes used in Southern and northern blotting are depicted in Figures 1 and 2. DNA or RNA is isolated from cells or tissues and then fractionated in a solid support (gel) in an electric field (electrophoresis). Following size separation, the DNA or RNA is transferred onto a membrane (generally nylon or nitrocellulose) and immobilized by exposure to ultraviolet radiation or heat. Membrane-bound DNA or RNA undergoes hybridization to a sequence homologous, labeled nucleic acid probe (usually prepared using cDNA or RNA). Detection of the probe depends on the labeling method employed. For example, with 32P-labeled probes the signal is detected and quantified using X-ray film (autoradiography). Probes are generated by several methods, including random-priming, nick-translation, and polymerase chain reaction (PCR). Sequences with only partial homology (e.g. species-specific cDNA or genomic DNA fragments) are used to prepare probes as well. Pros and ConsNorthern blotting remains the standard for detection and quantification of mRNA despite the development of more sensitive molecular techniques such as the reverse transcription-polymerase chain reaction (RT-PCR). It presents several advantages over newer techniques because it is a useful method for determining mRNA size and for identifying alternatively spliced transcripts and multigene family members. One advantage of Southern blotting is that it can be used to examine stretches of DNA such as the multikilobase restriction fragments produced by some restriction enzymes. For both Southern and northern blotting, the analysis of different genes via membrane stripping and reprobing also is an advantage over traditional PCR gel analysis techniques. A disadvantage of northern blotting is that it is the least sensitive of newer RNA analysis methods like PCR, gene microarrays, and ribonuclease protection assays (RPA). One disadvantage of Southern blotting is that it usually requires a relatively large quantity of high quality DNA. For this reason, PCR techniques are becoming widely applied to tasks for which Southern blotting was traditionally used (e.g. DNA fingerprinting and identification) because much less DNA is required and, in many cases, PCR techniques can be performed using partially degraded DNA. The use of radioisotopes in Southern and northern blotting also is considered a disadvantage from a health perspective, and the short half-life of phosphorus 32P (14 days) necessitates that the probe be prepared and used in an expeditious manner. Although it can be an advantage over traditional PCR gel analysis, reprobing of a Southern or northern membrane usually requires removal of the initial probe before hybridizing with a second probe. This process can be time consuming and can reduce subsequent DNA and RNA detection, a distinct disadvantage. PredictivitySouthern and northern blotting is used to better understand the molecular basis of disease. For example, northern blotting, which displays a high degree of correlation with RT-PCR, is used to monitor target gene expression or to identify biomarkers in isolated tissues and organs that results from a potential toxicological event or challenge. Genes of toxicological interest include proinflammatory cytokines, growth factors, cytochrome P450s, glutathione-S-transferases, cyclin dependent kinases, and cell cycle inhibitors. Relevance to HumansDNA and RNA underlie many aspects of human health, both in function and dysfunction. Southern and northern blotting will continue to aid in the identification of genetic events involved in neoplastic and non-neoplastic diseases, inherited disorders, genetic susceptibility, and predisposition to multigenic diseases. The accurate diagnosis and classification of acquired infectious diseases and neoplastic disease, as well as their prediction, prognosis, and therapeutic monitoring, will be aided by these technologies. Obtaining a detailed picture of how genes and other DNA sequences function together and interact with environmental factors ultimately will lead to the discovery of pathways involved in normal processes and in disease. Such knowledge will have a profound impact on the way disorders are diagnosed, treated, and prevented, and will bring about changes in clinical and public health practices. The characterization of (southern) and expression prof