An effective method for silver-staining DNA in large numbers of polyacrylamide gels

Silver-staining of nucleic acid has been used for various biological analyses, including polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis. A variety of methods have been described, but these methods are not that effective for staining more than a few PCR-SSCP gels, especially rapidly and with high sensitivity, because they include a number of time-consuming or hazardous manual steps that are often time dependent. Here we report a silver-staining method that can efficiently stain up to 14 gels at one time and with a detection limit of approximately 10 pg of DNA/mm², which is comparable to other methods.     

Negative Subtraction Hybridization: An efficient method to isolate large numbers of condition-specific cDNAs

Background  

The construction of cDNA libraries is a useful tool to understand gene expression in organisms under different conditions, but random sequencing of unbiased cDNA collections is laborious and can give rise to redundant EST collections.     

A flexible method to align large numbers of biological sequences

Summary A method for the alignment of two or more biological sequences is described. The method is a direct extension of the method of Taylor (1987) incorporating a consensus sequence approach and allows considerable freedom in the control of the clustering of the sequences. At one extreme this is equivalent to the earlier method (Taylor 1987), whereas at the other, the clustering approaches the binary method of Feng and Doolittle (1987). Such freedom allows the program to be adapted to particular problems, which has the important advantage of resulting in considerable savings in computer time, allowing very large problems to be tackled. Besides a detailed analysis of the alignment of the cytochrome c superfamily, the clustering and alignment of the PIR sequence data bank (3500 sequences approx.) is described.     

MRI contribution to the stereotactic management of cerebral tumours

Of 67 patients with cerebral tumours studied by MRI, 60 underwent stereotactic biopsy for histological diagnosis. The data from MRI were compared with those obtained from the CT scan with regard to the pathological diagnosis. The tumoural nature and extent of a lesion were better revealed by MRI. The single or multiple localization of the process was also seen better by MRI. Moreover, the sagittal-plane views shown by MRI provide much more accurate target placement and probe guiding for an orthogonal stereotactic approach. Finally, a post-biopsy MRI can show the biopsy site in relation to the tumour better.     

Verhulstian analysis of the growth of transplantable mammary tumours in sialoadenectomized mice

In this report we have analysed data published in 1989 by Inui et al. (Incidence of precancerous foci of mammary glands and growth rate of transplantable mammary cancers in sialoadenectomized mice. J. Natl Cancer Inst. 81, 1660) involving the effects of perturbation of the epidermal growth factor (EGF) status of mammary tumour-bearing mice on subsequent volumetric responses. Removal of an endogenous EGF stimulus by surgical ablation of the submaxillary glands, the major EGF-producing organ in mice, produced significantly slower growth of rodent mammary neoplasms, decreased success rate of transplantation, and an increase in the latent period before growth occurred. Administration of i.p. EGF (5 micrograms/mouse/day) to sialadectomized tumour-bearing mice would however, increase tumour growth rate. Data were analysed using the Verhulst equation which indicated that the observed effects on tumour volumetrics by either sialoadenectomy or EGF administration could be interpreted as being produced through paracrine pathways. The use of the Verhulstian analysis indicates that it is possible to analyse neoplastic responses and infer whether paracrine or autocrine pathways are involved.     

An alternative method to determine maximal accumulated O2 deficit in runners

An accepted measure of anaerobic capacity is the maximal O2 deficit. But it is not feasible to use O2 deficit if > or =10 submaximal runs are needed to extrapolate the O2 demand of high velocity running (Medb? et al. 1988). Recently, an alternative method to determine O2 deficit was proposed (Hill 1996) using only results of supramaximal cycle ergometer tests. The purpose of this study was to evaluate this alternative method with data from treadmill tests. Twenty-six runners ran at 95%, 100%, 105%, and 110% of their velocity at VO2max. Times to exhaustion, velocity, and accumulated oxygen uptake (VO2) from each individual's four tests were fit to the following equation using iterative nonlinear regression: accumulated VO2 = (O2 demand x velocity x time)-O2 deficit. The mean value s derived for O2 demand and O2 deficit were 0.198+/-0.031 ml x kg(-1) x m(-1) and 42+/-22 ml x kg(-1). SEE for the parameters were 0.007+/-0.007 ml x kg(-1) x m(-1) and 8+/-10 ml x kg(-1), respectively. Mean R2 was 0.998+/-0.003. It was concluded that O2 deficit can be determined from all-out treadmill tests without the need to perform submaximal tests.     

An alternative method in distinguishing cattle transferrin phenotypes

A modification of the method described by Kristjansson (1963) allows easier distinction of the components and position of every major cattle transferrin phenotype. The modification is based on increasing the percentage of starch (15%) and reducing the pH of the gel buffer to 6.8. In all the experiments, when a voltage of 350 was applied, a tray of ice was placed over the starch gel for the remainder of the electrophoresis. Different cattle transferrin phenotypes from our modified electrophoresis method are composed as follows: Type A, 4 bands; D₁, 4 bands; D₁D₂, 4 bands; D₂, 4 bands; E, 4 bands; AD₁, 6 bands; AD₂, 6 bands; AE, 8 bands and sometimes 9 bands; D₁E, 6 bands and sometimes 7 bands; and D₂E, 6 bands. The position of the fourth D band is distinctly different in D₁ vs. D₂ types.Journal Paper No. J-5937 of Iowa Agriculture and Home Economics Experiment Station, Ames. Project 1551.with a fellowship given by the Consejo National de Investigaciones Cientificas y Tecnicas de la Republica Argentina to work at the Department of Genetics, Iowa State University, Ames.     

An alternative, sensitive method to detect Helicobacter pylori DNA in feces

Background: Despite the high sensitivity and specificity of PCR, detection of Helicobacter pylori DNA in feces is still challenging. Fecal samples contain inhibitory molecules that can prevent amplification of the target DNA. Even by using specific DNA extraction kits for stools, monitoring of infection by analyzing stool samples remains problematic and endorses the need for improved diagnostic methods. Materials and Methods: The newly proposed method uses selective hybridization of target DNA with biotin‐labeled probes, followed by DNA isolation with streptavidin‐coated magnetic beads. After three washing steps, the purified DNA can be amplified immediately using conventional or quantitative PCR. In order to test this technique on biological samples, Mongolian gerbils were infected with H. pylori ATCC 43504 and fecal samples were analyzed on days 1, 4, and 10 post infection. Results: A detection limit of one bacterial cell per 100 mg stool sample was established, but only after removal of the magnetic beads from the target DNA by heating. This resulted in a 10‐fold increase of sensitivity compared to a commercially available stool DNA extraction kit. Analysis of fecal samples from infected gerbils demonstrated the presence of H. pylori DNA on each time point, while the uninfected animal remained negative. Conclusions: The proposed technique allows detection of very low quantities of H. pylori DNA in biological samples. In laboratory animal models, detailed monitoring of infection and complete clearance of infection can be demonstrated thanks to the low detection limit.     

A method for mini-immunoblots that allows screening of large numbers of samples

A method that allows the use of immunoblots as a screening test for large numbers of samples is described. Preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis is run to a separation distance of 2 cm and the samples are blotted onto nitrocellulose. One-millimeter-wide strips are cut and immobilized on double-sided tape. Adjacent strips are separated by plastic rods. After incubation with the first antibody, whole slides are processed together as in histology. The method has been successfully applied to the primary screening of hybridoma supernatants and is generally applicable in situations where large numbers of samples must be tested in a short time.     

Differential alternative splicing in brain regions of rats selected for aggressive behavior

Profiles of alternative mRNA isoforms have been determined in three brain regions of rats from an aggressive and a tame line selected for 74 generations. Among 2319 genes with alternatively spliced exons, approximately 84% were confirmed by analyzing public databases. Based on Gene Ontology-guided clustering of alternatively spliced genes, it has been found that the sample was enriched in synapse-specific genes (FDR < 10^–17). Patterns of gene expression in the brains of animals with genetically determined high or low aggression were more frequently found to differ in the use of alternatively spliced exons than in animals environmentally conditioned for increased or lowered propensity to aggression. For the Adcyap1r1 gene, five alternatively spliced mRNA isoforms have been represented differentially in aggressive animals. A detailed analysis of the gene that encodes glutamate ionotropic receptor NMDA type subunit 1 (Grin1) has confirmed significant differences in the levels of its alternatively spliced isoforms in certain brain regions of tame and aggressive rats. These differences may affect the behavior in rats genetically selected for aggression levels.     

An alternative method to the osmotic stressing polymers: the osmomanometer

Although stressing polymers have been widely and successfully used to determine the osmotic properties of solutes in aqueous media, the osmotic stress method presents some limitations. To overcome these drawbacks, an alternative and more direct method, which has been named the osmomanometer, is described in this letter. The osmotic pressure accessible by this method ranges typically from 1 to 30 kPa using a simple hydrostatic effect and can be extended to higher pressures by using pressurized gas. This method needs neither a pressure sensor nor calibration.