Gene analysis techniques最新文献

Presented here are the detailed methods employed in our laboratory for gene mapping and cytogenetic analyses in human beings, in the domestic cat, and in other mammalian species. Included in the procedures are: 1) establishment of primary fibroblast and lymphoid cell cultures; 2) heterologous cell fusion for production of rapidly proliferating cell hybrids; 3) cellular transformation of primary fibroblasts using an oncogenic retrovirus; 4) cell synchronization for high-resolution banding of prometaphase chromosomes; 5) chromosome-banding procedures, including G-banding, alkaline G-11, and Q-banding; and 6) in situ hybridization of radiolabeled molecular clones to metaphase chromosomes for regional gene localization.

Electroporation, the technique of electric field mediated gene transfer, was evaluated as a means of introducing and expressing genes into mouse Friend and human K562 erythroleukemic cells. Long-term (stable) gene expression in both Friend and K562 cells was measured using the recombinant plasmid Homer 6, which carries the aminoglycoside phosphotransferase (aph) gene as a selectable marker under the transcriptional control of the Moloney murine sarcoma virus long terminal repeat promoter/enhancer sequences. Parameters such as the DNA concentration, the initial field strength, the concentration of recipient cells, and the preselection expression time were examined to obtain optimal transfection frequencies. Short-term (transient) expression was also examined using the plasmid pLW4, which carries the chloramphenicol acetyltransferase gene under the transcriptional control of herpes simplex virus immediate early 5 gene promoter/enhancer sequences. Conditions that gave maximal stable transformation frequency were similar to those giving highest transient gene expression in the mouse and human erythroleukemic cell lines. Under optimal conditions, electroporation gave about ten times higher transfection frequencies and levels of transient expression for both types of cells when compared with the calcium phosphate technique. Because both Friend and K562 cells can be induced to differentiate in vitro, measurement of transient or stable expression levels for genes introduced into these cells may prove to be useful in the study of developmental regulation of genes from the erythroid pathway.

A solid-phase chemical degradation method for simultaneous sequencing of RNA and RNA fragments has been developed using Whatman DE 81 anion-exchange paper as the support. The approach involves the following operations: (1) immobilization of the 3′-end labeled RNAs or RNA fragments on DE 81 paper; (2) washing; (3) modification reactions; (4) washing; (5) sorting of the paper segments; (6) aniline reaction; (7) lyophilization; (8) desorption of the RNA by TEAB; and (9) lyophilization.

A simple method is described for the simultaneous isolation of both DNA and RNA from tissues and cultured cells obtainable in limited quantities only. The method is based on a suitable combination of steps designed for preparations of high molecular weight nucleic acids in cases when restricted amounts of tissues like small-sized and unique biopsies of tumors are available for studies of gene organization and expression. Using this protocol, undegraded total RNA suitable for Northern blot analysis and high molecular weight DNA for Southern blots was obtained from various sources (mammary and colon carcinomas, meningiomas, colonic and placental tissue, and several cell cultures).

A protocol is described for the growth and preparation of plasmid DNAs from small culture volumes (250 μl) and utilizing standard 96-well plates. Several hundred plasmids can be prepared simultaneously, yielding sufficient DNA for subsequent analysis by restriction digestion and gel electrophoresis. This protocol may be useful for rapid screening of clones arising in recombinant DNA work such as site-directed mutagenesis, oligonucleotide cassette cloning, deletion analysis, etc. The technique was initially developed to meet our requirement to provide large numbers of cosmid DNAs for restriction enzyme fingerprint analyses in genome mapping projects.

Quantitation of mRNA immobilized on nitrocellulose filters is an essential aspect of some studies in molecular biology. Hybridization of oligo(dT)₁₈ to the poly(A) tails of mRNA can be used to measure filter-bound mRNA and thus provides a basis for comparing abundance of specific mRNAs. Hybridization rate of ³²P-labeled oligo(dT)₁₈ in 0.75 M NaCl, 75 mM sodium citrate, pH 7 (5 × SSC) to immobilized RNA was maximal at 25°C. Filters were fully hybridized under these conditions within 1 hr when the oligo(dT)₁₈ concentration was 10 pmol/ml or higher. Salt dependence of the dissociation temperature (T_d) of oligo(dT)₁₈:RNA duplex on filters was described by the equation T_d = 42 − 20log₁₀[molar Na⁺] (°C). With stringent washing of the duplex (four 5-min washes in 2 × SSC at room temperature), oligo(dT)₁₈ gave no signal with plasmid DNA, rRNA, or tRNA. We have found that olig(dT)₁₈ can be used to normalize signal strengths rapidly and conveniently from total or oligo(dT)-selected eukaryotic RNA.

There has been a rapid increase in the number of available protein sequences derived from gene-sequence information. Computer-based sequence analysis of proteins is gaining in importance as an analytical tool. With the help of these analyses such sequences may be characterized and some insights gained into their probable role in the system. The principles involved in computer-based sequence analysis and some of the methods are discussed.

A method was developed for preparation of spheroplasts used for transfection with φX174 RF DNA. This method had a high-level competence and retained the competence for up to one year of storage in 7% DMSO at −70°C.

Medium-performance anion-exchange chromatography was applied to the purification of murine IgG class monoclonal antibodies from ascites fluid. The separations were performed under mild conditions at pH 8 using relatively low sodium-chloride concentrations. Recoveries for monoclonal antibodies of subclasses IgG1, IgG2a, and IgG2b were about 90%. The IgG preparations were free of other ascites fluid proteins.

A ligation assay was designed to determine if a ligation inhibitor is present in a Bio-Gel A-50-m column, the traditional tool used for separation of cDNA from free linkers during the λgtll cloning procedure. A new and simple electrophoresis method is also presented for performing this separation, which is rapid, efficient, and reduces the chance of carrying along a ligation inhibitor.