Detection of p53 point mutations by double-gradient, denaturing gradient gel electrophoresis

Genetic instability is a typical feature of tumor cells. This evidence has stimulated the development of rapid methods for detection of gene mutations. A new, improved protocol for denaturing gradient gel electrophoresis (DGGE), to screen for point mutations in genomic DNA, is reported: double gradient (DG) DGGE. In this technique, to the primary, denaturing gradient (typically 30-80% or 40-80% urea/formamide) a secondary gradient, colinear with the first, is superimposed: a porosity gradient (typically 6.5-12% polyacrylamide). The secondary gradient acts by recompacting smeared and diffuse bands of heteroduplexes, which are often indistinguishable from background fluorescence, and by augmenting the resolution between closely spaced homoduplex zones. This allows proper densitometric quantitation of the ratio of the two homoduplex bands. The reliability of this technique has been documented by detection of a number of mutations in exons 6 and 8 of the p53 gene which had escaped revelation by single-strand conformational polymorphism (SSCP) analysis. Additionally, the precise assessment of ratio of the doublet of homoduplex bands has allowed quantitation of the extent of p53 mutation in a mixed cell population extracted from a tumor specimen.     

A method for activity staining of peroxidase and beta-1,3-glucanase isozymes in polyacrylamide electrophoresis gels

Various mechanisms have been proposed for the aetiology of inflammation in ileal pouches following restorative proctocolectomy. It is proposed that many of these processes may be involved in the pathogenesis of ulcerative colitis, and therefore pouchitis may be used to study pathogenesis and treatment of inflammatory bowel disease in general and, in particular, ulcerative colitis.     

Isolation of canine prolactin by polyacrylamide gel electrophoresis

The electrophoretic mobility of prolactin obtained from canine pituitary extract was studied with the aid of polyacrylamide disc electrophoresis. Using a preparative gel electrophoretic system the immunoreactive material was purified on a quantitative scale which was then used to develop a homologous radioimmunoassay for canine prolactin. The radioimmunoassay system was able to detect prolactin in the plasma of dogs after the administration of agents which would be expected to affect prolactin secretion.     

Prenatal diagnosis of beta-thalassemia among Indians using denaturing gradient gel electrophoresis

We have offered first trimester prenatal diagnosis to 55 couples at risk for beta-thalassemia, originating from various parts of India, using polymerase chain reaction and denaturing gradient gel electrophoresis. Apart from the six common mutations, codon 30 (CAG-->CAA), Cap site +1 (A-->C), as well as three uncharacterized mutations were seen among the parents. In the majority of cases, the diagnosis was possible by scanning only one fragment (B) where most of the Indian mutations are situated. In 18 out of 55 cases, framework analysis could also have been used to offer prenatal diagnosis without characterizing the beta-thalassemia mutations. In the two cases where the mutations were uncharacterized, prenatal diagnosis was done only on the basis of the anomalous denaturing gradient gel electrophoresis patterns seen in the parents and in previously affected children. This is the first attempt of prenatal analysis using denaturing gradient gel electrophoresis in the extremely diverse Indian population where the profile of mutations has not yet been fully elucidated.     

Activity staining of protein inhibitors of proteases on gelatin-containing polyacrylamide gel electrophoresis

Stimulation of the supraorbital branch of the trigeminal nerve (SO) elicited eye blinks in the rabbit, but did not decrease the amplitude of visual cortical evoked potential from stimulation of the optic chiasm (OX). In addition, the SO stimulation neither induced an inhibitory postsynaptic potential (IPSP) in LGN cells, nor activated inhibitory interneurons in the thalamic reticular nucleus (TRN), which proved to mediate both recurrent inhibition and saccadic suppression in the dorsal lateral geniculate nucleus (LGN). All these indicate that there is no visual suppression in the rabbit LGN during blink reflex.     

Two-dimensional polyacrylamide gel electrophoresis map of bull seminal plasma proteins

A two-dimensional polyacrylamide gel electrophoresis map of bull seminal plasma proteins has been established. About 250 spots were detected after silver staining and polypeptides from 24 spots have been N-terminally sequenced. Major proteins already described in bull seminal plasma, like PDC-109 and aSFP, have been located on the map; proteins not yet reported in male reproductive tracts have been evidenced; for some polypeptides showing a previously unknown N-terminal sequence, structural similarities with proteins described in other organisms have been found. A reference map of seminal plasma proteins could be useful in relating protein pattern changes to physiopathological events influencing the reproductive sphere.     

Protein analysis of Strongyloides venezuelensis by two-dimensional polyacrylamide gel electrophoresis

Infective larvae, larvae in the lung and adult-stage worms in the small intestine of Strongyloides venezuelensis were analysed for protein by two-dimensional gel electrophoresis. The infective larvae were differentiated from the other two stages of parasite with 13 stage-specific spots, whereas the larvae in the lung and the adult-stage worms were identical to each other in spot patterns except for 6 spots.     

Changes in bacterial species composition in enrichment cultures with various dilutions of inoculum as monitored by denaturing gradient gel electrophoresis

Denaturing gradient gel electrophoresis revealed changes in the bacterial species obtained from enrichment cultures with different inoculum dilutions. This inoculum dilution enrichment approach may facilitate the detection and isolation of a greater number of bacterial species than traditional enrichment techniques.     

Optimized detection of DNA point mutations by double gradient denaturing gradient gel electrophoresis

Denaturing gradient gel electrophoresis displays the highest detection rate among mutation scanning methods. In classical denaturing gradient gel electrophoresis the denaturant gradient range and migration times vary for every amplicon to be scanned, greatly affecting the routine application of the method. As an alternative, we developed double gradient denaturing gradient gel electrophoresis where a gradient of pore size is superimposed over the denaturing one, allowing maintenance of the zone-sharpening effect even over prolonged time runs, and adoption of identical run time conditions for all fragments analyzed. Here double gradient denaturing gradient gel electrophoresis has been applied to the analysis of a number of point mutations and polymorphisms located in several exons of three different genes, the cystic fibrosis transmembrane conductance regulator, the beta-globin and the p53 genes.     

Detection by denaturing gradient gel electrophoresis of an Arg1689Cys mutation in a Chinese patient with mild hemophilia A

OBJECTIVE: To examine the ease of endotracheal intubation on the ground for various rescuer positions. METHODS: Six female and 18 male emergency medical technicians were asked to intubate a Laerdal Megacode Trainer placed on the ground. Rescuers assumed the following positions in random order: prone, sitting, kneeling at the mannequin's head, and straddling the chest. The authors measured times 1) for changing from mask ventilation to assuming intubation position and 2) from touching the laryngoscope to putting it down. Incidences of esophageal tube placement and clicks (possible tooth damage) were noted. The rescuers rated their satisfaction with each position on a six-point scale (1 = very good, 6 = insufficient). Total intubation times of the other three positions were compared with that for prone by rank order test for paired observations. Handling, esophageal positions, and clicks of the other three positions were compared with those for prone by sign test for paired observations. A Bonferroni correction (factor 12) was applied. RESULTS: Mean total intubation times (in seconds) were 11.8 +/- 3.3 for prone, 13.9 +/- 4.7 for sitting, 11.4 +/- 4.5 for kneeling, and 16.2 +/- 5.8 for straddling. The difference between straddling and prone was statistically significant (p < 0.005). For handling, the results were for prone 3.0 +/- 1.4, for sitting 3.1 +/- 1.1, for kneeling 2.2 +/- 0.6, and for straddling 2.8 +/- 1.4. Esophageal positions occurred for prone 1, for sitting 1, for kneeling 2, and for straddling 3. Clicks were counted for prone 2, for sitting 1, for kneeling 1, and for straddling 0. CONCLUSIONS: All tested positions provide satisfactory conditions for intubation on the ground. The straddling position requires statistically, but not clinically, significantly more time for intubation than does prone and may be an important backup position if access from behind the patient's head is impossible.     

Improvement of fragment and primer selection for mutation detection by denaturing gradient gel electrophoresis

Denaturing gradient gel electrophoresis (DGGE) is one of the most powerful methods for mutation detection currently available. For successful application the appropriate selection of PCR fragments and PCR primers is crucial. The sequence of interest should always be within the domain with the lowest melting temperature. When more than one melting domain is present the fragment is generally divided into several smaller ones. This, however, is not always necessary. We found that simple modifications of PCR fragments and primer sequences may substantially reduce the number of amplicons required. Furthermore, by plotting the (natural) melting curves of fragments without a GC-clamp, we could explain why fragments theoretically perfect for DGGE in practice failed to reveal mutations. Alternative fragment selection and the use of modified primers (addition of T/A or G/C tails) result in the detection of mutations that originally remained undetected. Our studies extend the utility of DGGE by using a minimum of PCR fragments and achieving a maximum of mutation detection.     

Correlation of plasma protein separation patterns obtained by two-dimensional polyacrylamide gel electrophoresis and by capillary electrophoresis

We used three different electrophoretic techniques for the analysis of human plasma proteins: (i) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), with sodium dodecyl sulfate (SDS) used only in slab gel electrophoresis; (ii) capillary isoelectric focusing (CIEF) with no denaturants; (iii) linear polyacrylamide (LPA)-filled capillary electrophoresis with SDS (SDS-CE). With technique (i), data on isoelectric point and molecular size of plasma proteins can be obtained. Techniques (ii) and (iii) are suited to obtain quantitative information on proteins. The separation principle used in technique (ii) is closely related to that used in the first dimension of technique (i), and that used in technique (iii) related to that in the second dimension of technique (i). Therefore, we could successfully correlate protein separation patterns obtained by 2-D PAGE and those obtained by capillary electrophoresis. The advantages of correlating data obtained by various electrophoretic techniques in the course of constructing a comprehensive database on human plasma proteins are discussed.     

Clinical implications of the types of cryoglobulins determined by two-dimensional polyacrylamide gel electrophoresis

BACKGROUND AND OBJECTIVE: Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a new method which can be used to study cryoprecipitates from the sera of cryoglobulinemic patients. It led to the identification of a new type of cryoprecipitate, tentatively named II-III, characterized by polyclonal IgG associated with a mixture of polyclonal and monoclonal IgM. Some discrepancies with the conventional classification of cryoglobulins were revealed. The association of particular clinical features with the classification of cryoglobulins by 2-D PAGE is examined. DESIGN AND METHODS: Sixty consecutive patients affected by cryoglobulinemic syndrome with mixed cryoglobulins were included in the study. All patients were evaluated for cutaneous, articular, hepatic, renal and nervous involvement. The washed cryoprecipitates were typed using both techniques: immunofixation electrophoresis (IFE) and 2-D PAGE. RESULTS: Sixteen (6 cases of type II and 10 of type III by IFE) of 60 cryoprecipitates (26.6%) appeared as type II-III by 2-D PAGE analysis. Nine cases were classified differently by IFE and 2-D PAGE. Mixed cryoglobulins of type II-III were not associated with a particular clinical pattern. Examining the clinical findings in the mono group (those with monoclonal IgM alone) and the poly group (those with polyclonal IgM alone or polyclonal and monoclonal IgM) we found clearly significant differences: more severe liver involvement in the poly group, and higher cryocrit and creatinine values, lower C4 level and more severe purpura in the mono group. INTERPRETATION AND CONCLUSIONS: Our results confirm the reliability of 2-D PAGE in characterizing cryoprecipitates. This sensitive method can demonstrate a higher number of monoclonal components, undetectable by IFE. Type II-III cryoglobulins are not associated with a particular clinical pattern. The presence or absence of polyclonal IgM in mixed cryoglobulins seems to be correlated with some clinical findings.