Gene analysis techniques最新文献

A method is presented that facilitates the identification of cDNA clones corresponding to the polyadenylated 3′ end of mRNA. It is based on the use of a poly dT probe that is synthesized by homopolymer extension of commercially available oligo dT. The method is shown to work in Southern blot analysis of plasmid preparations and in situ with colonies.

The genetic locus and primary structure of the human immunodeficiency virus (HIV) protease was determined by comparing the data of protein analyses with the published data of the gene analysis. The complete sequence of HIV-1 and HIV-2 protease was synthesized by solid-phase peptide synthesis. The synthetic protease was capable of accurately cleaving synthetic peptide substrates corresponding to known cleavage sites in gag polyproteins of HIV-1, HIV-2, and murine leukemia virus. The chemical synthesis of protease confirms the DNA sequence and provides a means of rapidly producing active protease in substantial quantities for biochemical and physical studies.

RNA binding proteins partially constitute theribonucleoprotein or protein machinery for RNA processing (splicing, polyadenylation and 3′ end formation), transport, and storage. We have devised a novel method for the detection of RNA binding proteins in vitro. The template for transcription is a cloned Drosophila melanogaster 5S rRNA gene. The method is a two-dimensional gel analysis involving: in vitro transcription of ³²P-labeled 5S rRNA using a cellular S-100; resolution of labeled RNA protein complexes from unbound RNA on a first-dimension mobility shift gel; cross-linking of RNA to protein in gel by ultraviolet irradiation; degradation of the RNA by RNase A and Tl; and analysis of ³²P-protein patterns on a second-dimension discontinuous SDS gel by autoradiography. The pattern of proteins associated with ³²P-5S rRNA is obtained by covalent transfer of ³²P-nucleotides from RNA to the proteins with which the RNA was bound. This method could be useful in the analysis of RNA maturation and processing pathways.

We wish to report the initial characterization of a recombinant clone containing the BamHI methylase gene. Genomic chromosomal DNA purified from Bacillus amyloliquefaciens was partially cleaved with HindIII, fractionated by size, and cloned into pSP64. Plasmid DNA from this library was challenged with BamHI endonuclease and transformed into Escherichia coli HB101. A recombinant plasmid pBamM6.5 and a subclone pBamM2.5 were shown to contain the BamHI methylase gene based on three independent observations. Both plasmids were found to be resistant to BamHI endonuclease cleavage, and chromosomal DNA isolated from E. coli HB101 cells harboring either of the plasmids pBamM6.5 or pBamM2.5 was resistant to cleavage by BamHI endonuclease. In addition, DNA isolated from lambda phage passaged through E. coli HB101 containing either plasmid was also resistant to BamHI cleavage. Expression of the BamHI methylase gene is dependent on orientation in pSP64. In these clones preliminary evidence indicates that methylase gene expression may be under the direction of the plasmid encoded LacZ promoter.

A single buffer consisting primarily of potassium and glutamate is shown to be suitable for activity of almost all restriction endonucleases, DNA methylases, and many other DNA-modifying enzymes. The composition of this buffer differs substantially from buffers currently in use for these enzymes.

This paper describes a method for isolation of DNA from blood samples involving a rapid chemical disintegration of proteins with 8 M urea and with a minimum of exposure to phenol. The DNA is further desalted and purified on Sephadex G-25 prepacked disposable columns. DNA isolated in this way was pure enough to be immediately cleaved by restriction enzymes.

A technique for the rapid isolation of mitochondrial DNA (mtDNA) from animal tissues is described that eliminates the time-consuming separation of nuclear and mtDNAs using cesium chloride gradient ultracentrifugation. The procedure utilizes digestion of the nuclear DNA with DNase, after which lysis of mitochondria and subsequent extraction of proteins results in relatively pure mtDNA. Up to 5 μg of mtDNA per gram of liver tissue resulted, a suitable yield for five digests with restriction enzymes and staining with ethidium bromide.

We present a unified algorithm to analyze distances between short oligomers in large collections of nucleic acids and protein sequences (DISTANP). This extended version of DISTAN methodology not only permits analysis of distances between selected pairs of oligomers, but also allows a user to analyze distances between groups of residues (such as acidic and hydrophobic amino acids). This capacity allows differentiation of sequence properties of known functional domains in nucleic acids and proteins.

A simple and efficient method for determining restriction fragment length polymorphism types on large numbers of individuals using small samples of peripheral blood or sperm cells is described. Whole cells embedded in low gelling/melting temperature agarose were treated with a series of enzyme, detergent, and washing steps to release high molecular weight DNA that was then digested with standard restriction enzymes such as EcoRI and PstI, electrophoresed, blotted, and probed as in normal Southern analyses. The technique should be readily adaptable to any application requiring DNA from small numbers of cells for Southern analyses or pulsed field gel electrophoresis.

A rapid and efficient procedure for purifying bacteriophage λ DNA is described. This small-scale purification involves isolation of bacteriophage particles on cesium chloride gradients. Using an Airfuge ultracentrifuge, the centrifugation step can be readily achieved in 90 minutes. The method allows a 1-day purification of up to 12 independent λ DNA (20–40 μg each). The recovered DNA, essentially devoid of RNA and DNA contaminants, is efficiently cut by restriction endonucleases and can serve as starting material for the ligation of DNA fragments in other cloning vehicles.