Gene amplification and analysis最新文献

Among the various restriction sites present on a DNA molecule, the restriction endonucleases prefer specific ones. This site preference may be an inherent property of the restriction endonucleases or may reflect the complexities inherent in the DNA molecule. The site preference of restriction endonucleases can be amplified by the use of intercalators that bind to DNA. This can lead to the production of large and partially cleaved DNA fragments. General protein inhibitors that react with sulfhydryl groups can affect the activities of some restriction endonucleases. This can result in the formation of partially digested DNA fragments. Another approach leading to the formation of large DNA fragments involves base substitution or modification of DNA molecules. New restriction sites can be exposed by relaxing the specificity of some restriction endonucleases. Under conditions of relaxed specificity, the recognition sequence shrinks to the core sequence, which is usually two nucleotides shorter than the normal recognition sequence. When the core restriction sequences are unmasked by relaxation of restriction-endonuclease specificity, the normal restriction sequences inaccessible in some DNAs can be exposed by the prevention of DNA modification. All manipulations described here lead to the formation of DNA fragments that are different (large or new) from normal restriction-endonuclease digestion products. These DNA fragments have potential applications in the mapping of DNA, gene-cloning experiments, and genetic experiments on deletion or substitution.

S1 nuclease has found many uses in the analysis of the structure of nucleic acids, and more new applications, such as the mapping of splice points of early mRNAs in SV40, will undoubtedly be found.

In recent years, there has been a growing appreciation of the potential applications of 5'-32P-end-labeled mRNA, not only for screening recombinant clones and mapping gene structure, but also for revealing possible nucleotide sequence and structural signals within mRNA molecules themselves, which may be important for eukaryotic mRNA processing and turnover and for controlling differential rates of translational initiation. Three major problems, however, have retarded progress in this area, lack of methods for efficient and reproducible removal of m7G5ppp5'-cap structures, which maintain the integrity of an RNA molecule; inability to generate a sufficient amount of labeled mRNA, owing to the limited availability of most pure mRNA species; and the frequent problem of RNA degradation during in vitro end-labeling owing to RNAse contamination. The procedures presented here permit one to decap and label minute quantities of mRNA, effectively. Tobacco acid pyrophosphatase is relatively efficient in removing cap structures from even nanogram quantities of available mRNA, and enough radioactivity can be easily generated from minute amounts ofintact mRNA with very high-specific-activity [gamma-32P]ATP and the inhibition of ribonuclease contamination with diethylpyrocarbonate. These procedures can be modified and applied to almost any other type of RNA molecule as well. In Section III of this volume, we explore in detail how effectively 5'-end-labeled mRNA can be used not only for nucleotide sequence analysis, but also for mapping mRNA secondary structure.