Gene analysis techniques最新文献

A procedure to measure chloramphenicol acetyl transferase (CAT) activity by reverse-phase high-performance liquid chromotography is described. The antibiotic as well as the acetylated derivatives are well resolved on a Superspher RP-18 column using equal parts of acetonitrile and 10 mM sodium acetate (ph 5.0) as a solvent. Under these conditions, less than 100 pmol of each derivative can be easily detected within 10 minutes, and no radioactive chloramphenicol is needed. The present procedure has been used to measure the activity of the enzyme in extracts of chicken fibroblast transfected with the recombinant plasmid pSV2-cat containing the CAT gene.

We have investigated the use of in vitro expression as a quick and convenient means of screening large numbers of interferon (IFN) analogs generated using in vitro mutagenesis. The IFN-α1 mRNA generated from DNA template using SP6 RNA polymerase is efficiently translated in rabbit reticulocyte lysate (RRL). The antiviral specific activity of this RRL-synthesized IFN-α1 is equivalent to the yeast-synthesized protein. In contrast with the yeast-expression system, where some IFN-α analogs are poorly expressed, all analogs tested were well expressed in RRL.

We have developed a method called oligo-scanning mutagenesis that uses oligonucleotides to mutate up to 12 contiguous bases in a single step. Some advantages of this procedure are that the position and sequence of the replacement mutations are completely specified by the investigator, and combinations of mutations can easily be generated. The technique uses a gapped substrate and the Escherichia coli dam methylation error-correcting mechanism to increase the yield of mutants.

Pulsed field gel electrophoresis (PFGE) is a powerful new tool for genetic analysis that can be applied to a variety of problems concerning genome structure and organization. This technique uses an agarose gel matrix to separate DNA molecules in a size range from 40 kb to 2,000 kb, molecules far larger than the maximum separable using standard agarose gel electrophoresis. The PFGE method can be used to separate the intact chromosomes from lower eukaryotes or to separate very large DNA fragments from higher eukaryotes generated by digestion with restriction endonucleases whose cleavage sites are rare. This paper describes the use of PFGE for construction of long-range restriction maps in the human genome and includes detailed methods for all steps. A pulsed field gel device that utilizes a rotating platform for altering the applied electric field is also described. Map construction is illustrated using a cloned DNA fragment (D3S2) from human chromosome 3. Several technical problems specific for mammalian genomes are discussed.

This paper describes methods that are commonly used for performing mRNA in situ hybridizations. Each stage of the procedure has been analyzed to identify the parameters that most significantly affect the final cell morphology and sensitivity of the system. We have identified key elements of the procedure as the fixation employed, the type of polynucleotide probe and label chosen, and the detection system used. By optimizing these critical components, we have developed a procedure for performing mRNA in situ hybridizations that takes 2–4 hours and has a sensitivity of 1–10 molecules of mRNA per cell. This system has been used to detect levels of oncogene expression in normal bone marrow and peripheral blood. It is possible to detect the expression of three oncogenes (c-myc, c-sis, and c-abl) simultaneously in a small population of cells from the peripheral blood of leukemic patients.

A method is described for cloning synthetic oligodeoxynucleotides, which can theoretically be of any length. The method requires only a single oligodeoxynucleotide strand and a vector with two unique restriction sites, one of which is for an enzyme that generates 3′ protruding ends. A mixture of unpurified oligonucleotides containing a wild-type genetic regulatory sequence of the Escherichia coli gnd gene and two mutations of it was cloned into a plasmid carrying a gnd-lacZ protein fusion. Individual cloned oligonucleotides were readily identified by direct DNA sequencing of plasmid templates. The method is rapid, efficient, and has application to gene synthesis and site-directed mutagenesis.

A method is described for the isolation of high molecular weight DNA in solution using the principles that have allowed electrophoresis of chromosome-sized DNA in pulse field gradient electrophoresis. Stationary phase yeast cells are converted to spheroplasts by the action of zymolyase in 1 M sorbitol. In the presence of EDTA and sodium lauroyl sarcosinate, proteins are digested with proteinase K. DNA is extracted with phenol and chloroform, and high molecular weight DNA is collected by ethanol precipitation. RNA is removed by RNase digestion of the redissolved pellet, and RNase is removed by chloroform extraction followed by a second ethanol precipitation. The method is rapid and gives a high yield of DNA that is readily digestible by restriction endonucleases.

Computer-assisted sequence analysis was applied to detect the most apparent nonrandom sequence motifs in eukaryotic introns. We describe in detail a method, which we call distance analysis, that we applied to the extensive study of 405 eukaryotic intron sequences. We observed very strong two-base periodicities for almost all tetranucleotides that are tandem repeats of nonhomopolymeric dinucleotides (the exception was GCGC and CGCG). We also observed, by using a fixed-point alignment method, that these periodic sequence motifs belong to large clusters of dinucleotides repeated tandemly as many as 15–35 times, which corresponds to the cluster lengths of 30–70 bases. We did not observe two-base periodicity of tetranucleotides in the collections of either 262 spliced eukaryotic exons or 107 bacterial genes. Instead, these sequences displayed strong three-base periodicity of some other tetranucleotides. These findings suggest that introns and exons display distinct sequence properties that can be used for mapping purposes.