An Improvement on Labelled Messenger RNA Preparation for DNA-RNA Hybridisation

Abstract

An improved method for the removal of DNA and phenol residues after phenol extraction in m-RNA preparation has been developed. This involves repeated freeze-thaws of the aqueous layer under centrifugal force. As a model assay, five minutes pulse-labelled AcI47 late m-RNA is prepared and hybridised to the r-strain ADNA. After the centrifugation treatment, non-specific hybridisation is reduced to one-tenth of the untreated sample and the background with respect to input radioactivity for hybridisation is reduced to 0.3% This low background m-RNA preparation is important especially in dealing with minor class m-RNA.     

Synthesis of nascent prothrombin and albumin in a heterologous system using rat liver messenger RNA purified in oligo (dT)-cellulose.

Highly active m-RNA was prepared by phenol extraction of rat liver polysomes followed by oligo (dT)-cellulose chromatography. This m-RNA preparation stimulated total protein synthesis in rabbit reticulocyte lysates and in wheat germ extracts. Nascent prothrombin and albumin synthesized in the reticulocyte system programmed with this m-RNA were precipitated with specific antibodies and identified by their electrophoretic mobilities on SDS-acrylamide gels.     

The biological activity of bacteriophage DNA, prepared by the cationic detergent dilution technique

The preparation of phage lambda DNA infecting E. coli K 12 with cationic detergent is described. This DNA infects E. coli spheroblasts with the same efficiency as DNA prepared by phenol methods.     

Methodology: simplified preparation of a DNA ladder using PCR

Serving as a DNA molecular weight standard, the DNA ladder has been widely used in molecular biology applications. We developed a simple method for the preparation of a DNA marker, which involves designing primers to amplify 100- to 1000-bp DNA fragments using lambda DNA as a template for polymerase chain reaction, followed by extraction with phenol/chloroform, precipitation with ethanol and mixing. Fragments of 100- to 1000-bp DNA were successfully amplified; the sequences showed 100% identity with lambda DNA. This prepared DNA marker displayed clear bands, indicating that it can be used for molecular studies.     

Preparation of bacteriophage lambda DNA using the TL-100 ultracentrifuge

A procedure for the preparation of DNA from bacteriophage lambda is described, using the Beckman TL-100 bench-top ultracentrifuge. The procedure involves growth of phage in agar plates, precipitation with polyethylene glycol, and a single centrifugation in cesium chloride under conditions that disrupt the phage coat. The method avoids the use of enzymes, ion exchange resins, and phenol. It can be completed in less than a day. The resulting DNA is of good purity and is easily cuttable by restriction enzymes.     

A simple and efficient procedure for the isolation of high-quality phage lambda DNA using a DEAE-cellulose column

A simple and rapid procedure for purifying large quantities of bacteriophage lambda particles and DNA is described. The procedure involves DEAE-cellulose column chromatography of the phage particles and elution of the phage particles from the column with a low-ionic-strength buffer. The resulting phage were well separated from RNA, DNA, and proteins derived from Escherichia coli host cells. The lambda DNA was prepared from the purified phage particles by the conventional method of phenol extraction and ethanol precipitation. This procedure did not use nucleases, proteases, detergents, or CsCl density gradient centrifugation. The lambda DNA obtained by this method was equivalent in purity to the material prepared by CsCl density gradient centrifugation and amenable to restriction enzyme digestion, ligation, radiolabeling, and double-stranded DNA sequencing. A detailed protocol is described for obtaining 0.5 to 1.0 mg DNA from a 1-liter liquid lysate in less than 5 h. This procedure is simple, inexpensive, and timesaving, and is particularly suitable for large-scale isolation of lambda DNA.     

A bacteriophage lambda DNA purification procedure suitable for the analysis of DNA from either large or multiple small lysates

A method for the efficient preparation of high quality bacteriophage lambda DNA from cleared lysates is described. Advantages of the method include high DNA yields (typically around 0.8 micrograms of DNA/1 ml of cleared lysate), speed of processing (approximately 2 h from lysate to DNA), economy, and the absence of any requirement for phenol or chloroform extractions. The technique involves the concentration of phage particles by standard polyethylene glycol precipitation followed by enzymatic treatment to remove contaminating RNA and DNA. Phage particles are then lysed with sodium dodecyl sulfate (SDS) at elevated pH and temperature. Contaminating protein/SDS complexes are rendered insoluble by the addition of potassium acetate and removed by centrifugation. The quality of the resultant DNA is comparable to that prepared by cesium chloride banding for all standard molecular biological purposes providing that spermidine is included in all restriction endonucleases digestions.     

PCR preparation of DNA inserts from lambda and plasmid vectors for RFLP mapping

A simplified method is described for preparing insert DNA for labelling reactions to be used in Southern hybridization. This method works with sequences cloned into both plasmid and lambda phage, and eliminates many of the steps leading to the labelling reaction. Small quantities of hostE. coli or lambda phage carrying a probe sequence are lysed and amplified via the polymerase chain reaction using standard sequencing primers. Unincorporated nucleotides are removed by ethanol precipitation or gel purification and insert DNA is ready for radio-labelling. This method reduces the time and expense associated with conventional insert preparation, and greatly simplifies the use of sequences cloned into lambda phage.     

An improved technique for the efficient construction of gene libraries by partial filling-in of cohesive ends

For the preparation of gene libraries, DNA from lambda EMBL3 phage was digested with SalI and EcoRI, and the cohesive ends partially filled-in by addition of dTTP, dCTP and Klenow fragment of DNA polymerase I (PolIk). Genomic DNA was cleaved partially with Sau3A and subsequently incubated with dATP, dGTP and PolIk. The phage and genomic DNAs were then mixed and ligated. The recombinant DNAs were packaged in vitro. The efficiency of packaging was 10(5)-10(6) of infectious phage lambda particles per microgram of the genomic DNA (as compared to approx. 10(7) per microgram for the wild-type lambda DNA). This procedure is very rapid and requires only microgram quantities of genomic DNA for preparing an entire gene library. The other important advantage is that multiple independent insertions of genomic DNA cannot occur in a single recombinant phage and self-ligation of phage DNA is blocked. It is also applicable for other SalI-containing vectors.     

Large-Scale Production of Lambda Bacteriophage and Purified Lambda Deoxyribonucleic Acid

Large amounts of a heat-inducible phage lambda mutant (lambdaCI857) may be obtained under standardized conditions. The phage is harvested by simple polyethylene glycol (C 6000) precipitation and purified by CsCl density gradient banding. Deoxyribonucleic acid (DNA) is extracted by cold phenol and purified by sucrose density gradient sedimentation to yield a homogenous population of unbroken lambda DNA molecules.     

A rapid method for isolation of double stranded RNA

A rapid and simple method for the isolation and purification of dsRNA is presented. The crucial step of this method is the extraction of proteins and DNA with acid phenol. After the extraction, only RNA is left in the aqueous phase. ssRNA contamination of the RNA preparation can be greatly reduced when ammonium sulfate is present during the extraction.     

A Rapid Method for Isolation of Double Stranded RNA

A rapid and simple method for the isolation and purification of dsRNA is presented. The crucial step of this method is the extraction of proteins and DNA with acid phenol. After the extraction, only RNA is left in the aquaeous phase. ssRNA contamination of the RNA preparation can be greatly reduced when ammonium sulfate is present during the extraction.     

Location and organization of the dimethylphenol catabolic genes of Pseudomonas CF600

Summary The gene organization of the phenol catabolic pathway of Pseudomonas CF600 has been investigated. This strain can grow on phenol and some methylated phenols by virtue of an inducible phenol hydroxylase and meta-cleavage pathway enzymes. The genes coding for these enzymes are located on pVI150, an IncP-2 degradative mega plasmid of this strain. Twenty-three kilobases of contiguous DNA were isolated from lambda libraries constructed from strains harbouring wild type and Tn5 insertion mutants of pV1150. A 19.9 kb region of this DNA has been identified which encodes all the catabolic genes of the pathway. Using transposon mutagenesis, polypeptide analysis and expression of subfragments of DNA, the genes encoding the first four enzymatic steps of the pathway have been individually mapped and found to lie adjacent to each other. The order of these genes is the same as that for isofunctional genes of TOL plasmid pWWO and plasmid NAH7.     

Atomic force microscopy imaging of double stranded DNA and RNA.

A procedure for imaging long DNA and double stranded RNA (dsRNA) molecules using Atomic Force Microscopy (AFM) is described. Stable binding of double stranded DNA molecules to the flat mica surface is achieved by chemical modification of freshly cleaved mica under mild conditions with 3-aminopropyltriethoxy silane. We have obtained striking images of intact lambda DNA, Hind III restriction fragments of lambda DNA and dsRNA from reovirus. These images are stable under repeated scanning and measured contour lengths are accurate to within a few percent. This procedure leads to strong DNA attachment, allowing imaging under water. The widths of the DNA images lie in the range of 20 to 80nm for data obtained in air with commercially available probes. The work demonstrates that AFM is now a routine tool for simple measurements such as a length distribution. Improvement of substrate and sample preparation methods are needed to achieve yet higher resolution.     

Isolation of lambda phage DNA by hydroxylapatite chromatography

A simple and rapid (1 day) method for preparation of lambda phage DNA was proposed. The method included two main steps: (a) growth and lysis of bacteria containing lambda phage and (b) purification of lambda phage DNA by hydroxylapatite chromatography. The phage DNA prepared by this method was intact and free of RNA, proteins, and bacterial DNA.     

Atomic Force Microscopy Imaging of Double Stranded DNA and RNA

Abstract

A procedure for imaging long DNA and double stranded RNA (dsRNA) molecules using Atomic Force Microscopy (AFM) is described. Stable binding of double stranded DNA molecules to the flat mica surface is achieved by chemical modification of freshly cleaved mica under mild conditions with 3-aminopropyltriethoxy silane. We have obtained striking images of intact lambda DNA, Hind III restriction fragments of lambda DNA and dsRNA from reovirus. These images are stable under repeated scanning and measured contour lengths are accurate to within a few percent. This procedure leads to strong DNA attachment, allowing imaging under water. The widths of the DNA images lie in the range of 20 to 80nm for data obtained in air with commercially available probes. The work demonstrates that AFM is now a routine tool for simple measurements such as a length distribution. Improvement of substrate and sample preparation methods are needed to achieve yet higher resolution.     

A simple purification procedure for lambda gt bacteriophage DNA with hybridization size screening for isolation of longest length cDNA clones

An improved procedure for isolating lambda DNA and screening lambda gt10 or lambda gt11 libraries is described. Recombinant lambda gt11 bacteriophage particles (150,000) were amplified on three agarose plates (50,000 per plate) with Escherichia coli Y1090 as plating bacteria. After confluent lysis, recombinant bacteriophage was extracted with SM buffer. Bacterial debris was removed by centrifugation. A small aliquot of amplified lambda gt11 bacteriophage was kept to rescreen the bacteriophage, should a large or full-length clone be found to be present, after analysis of the size of the cDNA inserts. The major portion of the bacteriophage particles was purified by treatment with equilibrated DEAE-cellulose, pH 7.5. Purified phage particles were precipitated with polyethylene glycol from the DEAE supernatant and extracted with phenol, phenol-chloroform, and chloroform. Such lambda gt11 DNA was readily digested with EcoRI. Liberated insert cDNA was separated on 1.2% agarose gels, transferred onto a nylon membrane, and hybridized with an alkaline phosphatase cDNA probe in an iterative procedure that allows isolation of the largest cDNA clones present in the library. We have used this procedure to isolate a full-length alkaline phosphatase cDNA. The method is quick, reliable, and less costly than conventional procedures for the isolation of full-length cDNAs.     

New Method for Large-Scale Preparation of Covalently Closed λ DNA Molecules

A combination of mutations in bacteriophage lambda and its host Escherichia coli K-12 provides a convenient system for the isolation of large quantities of covalently closed circular DNA molecules. We describe two procedures for the large scale preparation of lambda DNA in the duplex circular form.     

Selective inhibition of Escherichia coli recBC activities by plasmid-encoded GamS function of phage lambda

The gam locus of bacteriophage lambda encompasses two coding sequences with the same reading frame and translational stop, one corresponding to an Mr 11646 polypeptide (gamS gene), the other to an Mr 16349 polypeptide (gamL gene). A DNA segment encoding gamS but not gamL was placed under lambda pR promoter control (regulated by the cIts857-coded repressor) on a multicopy plasmid, and an insertion mutation (gamS201) was constructed. Expression of gamS+, but not gamS201, inhibited Escherichia coli RecBC nuclease in vivo; the criteria were inhibition of chromosomal DNA degradation after UV irradiation and plating of T4 gene 2- phages. The recB+ C+ bacteria expressing gamS+ were completely or partially similar to recC- mutants with respect to certain phenotypes: defective plating of phages P1 and P2, ability to plate (in a recA- background) lambda red- gam- phages, reduced resistance to UV irradiation, defective SOS induction, decreased colony-forming ability.