摘要:

AbstractAlusequences are present in humans in excess of 500 000 copies per haploid genome and represent the largest family of short interspersed repetitive elements (SINEs). These mobile genetic elements are ancestrally derived from the 7SL RNA gene and move throughout the genomes of primates by retroposition. PolymorphicAluinsertions have proven to be useful for population studies, paternity determinations and forensic applications. Additionally, a simple polymerase chain reaction (PCR)‐based assay has been established to examine these polymorphisms. In the present study, we have applied the technique of multiplex polymerase chain reaction to theAlupolymorphic system. Duplex and triplex PCR reactions were performed for the analysis of five differentAlupolymorphic loci in different combinations. This study represents a starting point for further experimentation to improve and eventually optimizeAlumultiplex PC

ISSN:0173-0835    

DOI:10.1002/elps.1150191402

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractIn genetic toxicology, the main fields of applications of the polymerase chain reaction (PCR) with subsequent electrophoretic characterization of amplificates include genotyping polymorphisms in the xenobiotic metabolism and mutant analysis. To assess the role of the individual sets of biotransformation enzymes for the internal dose resulting from xenobiotic exposure, we investigated blood samples from 69 healthy donors for the occurrence of known genetic polymorphisms in the xenobiotic metabolizing enzymesN‐acetylaminotransfrase II (NAT2), glutathione‐S‐transferase (GST) μ and θ, and several cytochromes P450 (CYP), namelyCYP1A1,CYP2E1andCYP2A6. Using single strand conformation polymorphism (SSCP) analysis, five known single base substitutions located in the middle portion of 144 bp amplificates comprising exons 7 and 8 of the human hypoxanthine guanine phosphoribosyl transferase (hprt) cDNA, were clearly distinguished from wild type and from each other. Biomagnetic strand separation assigned the slower migrating single strand bands to the biotinylated sense

ISSN:0173-0835    

DOI:10.1002/elps.1150191403

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractBecause of the repetitive nature of the sequence, when titrating a G,A‐rich, triple helix‐forming oligonucleotide (TFO) with increasing concentrations of target duplex in order to obtain the dissociation constant of the complex, a duplex is sometimes first generated at intermediate concentrations of the target. This duplex is based on an imperfect Watson‐Crick pairing of the TFO to the pyrimidine‐rich strand of the target. An explanation is proposed for this duplex being obtained only in a certain domain of the titration range, before the triple helix becomes pred

ISSN:0173-0835    

DOI:10.1002/elps.1150191404

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractTesting different theories of concerted evolution experimentally has been hampered mainly due to the lack of appropriate model systems and technical limitations. In this study, we employed a denaturing gradient gel electrophoresis (DGGE) approach for the display and definition of nucleotide variations in the second internal transcribed spacer (ITS‐2) of ribosomal DNA (rDNA) of the parasitic nematode,Haemonchus contortus. The ITS‐2 was amplified from individual adult nematodes by PCR and subjected to DGGE. Of the 94 individuals (representing nine different populations) analysed, 13 different DGGE profiles were displayed. Eighteen bands representing those profiles were excised and sequenced. Sequencing defined 13 different types of ITS‐2 with 12 nucleotide variations (4 transitions, 5 transversions, 1 insertion and 2 deletions) which could be related to particular positions of the predicted secondary structure for the ITS‐2 pre‐rRNA. The results showed that individuals of interbreeding populations ofH. contortuscan have rDNA arrays that are partially or fully homogenised for different sequence variants (despite interindividual variation), suggesting that the homogenisation process is driven mainly by intrachromosomal exchange. The findings also demonstrated the capacity of the DGGE‐sequencing strategy to quantify the frequency of ITS‐2 sequence types within individual nematodes from different populations without the need for cloning or Southern blot procedures. This has important implications for studying the mechanisms of sequence homogenisation in rDNA and pre‐rRNA processing as well as for elucidating speciation events and population differentation at the

ISSN:0173-0835    

DOI:10.1002/elps.1150191405

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractApplied DNA typing in medico‐legal investigations, in criminalistic practice, and in paternity cases often relies on high inclusion and exclusion probabilities. For that reason, the short autosomal tandem repeat locus D8D306 was validated for forensic use and incorporated into a nonoverlapping multiplex reaction with HUMDHFRP2 and HUMCD4. The allele frequencies of D8S306 in four different regions of Germany (n= 1220 alleles) were determined for use in a population database; the allele distributions did not significantly deviate from each other. The hererozygosity of D8S306 is 83%, expected exclusion chance in stain cases is 96% (paternity cases: 69%), the lowest amount of sucessfully amplified DNA was 30 pg. The alleles are in Hardy‐Weinberg equilibrium (exact te

ISSN:0173-0835    

DOI:10.1002/elps.1150191406

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractWe have recently shown that a few nanograms of protein separated by electrophoresis in sodium dodecyl sulfate‐polyacrylamide gels can be detected by reverse‐staining, exploiting the precipitation reaction between zinc(II) and imidazole. Modifications of this method have also been generated to detect gelisolated nucleic acids and bacterial glycolipids. Because there is no recourse to chemical modifiers, the reverse‐staining technique has been valuable when micropreparing these biomacromolecules for later use or characterization. The mechanism underlying the reverse‐staining effect, however, remains incompletely understood and this has prevented a further generalization of the technique. Here, we have conducted physicochemical experiments and identified zinc imidazolate (ZnIm2) as the main component of the precipitate that forms along the surface of zinc‐imidazole reverse‐stained gels. Many staining effects observed when gels containing electrophoretically separated biopolymers are subjected to zinc‐imidazole stains have been rationalized. The reverse‐staining method has been vastly generalized, now allowing the detection of proteins and glycolipids as well as complexes of these macromolecules in native gels. We demonstrate the application of the reverse‐staining technique in situations where Coomassie blue or silver staining was inappropriate or failed to produce detection of the species of interest. The present generalization of the reverse‐staining method facilitated the characterization of biomacromolecular interaction partners in mixtures of bacterial glycolip

ISSN:0173-0835    

DOI:10.1002/elps.1150191407

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractThe fluorogenic dye 2‐methoxy‐2,4‐diphenyl‐3(2H)‐furanone (MDPF) has been used for the detection of total protein patterns on polyvinylidene difluoride (PVDF) membranes. Fluorescent staining of protein bands on membranes with this covalent dye is completed in 20 min. Wet membranes are translucent, allowing protein visualization by transillumination with ultraviolet light. The resulting images can be recorded using Polaroid film or a charge‐coupled device camera. Electrophoretic bands containing 5–10 ng of protein can be detected on the MDPF‐stained Western blot. When proteins are directly transferred to the membrane using a slot blotting device, as little as 0.5 ng of protein can be detected. Previous visualization of protein bands on sodium dodecyl sulfate‐polyacrylamide gels with the noncovalent fluorescent dye Nile red (Albaet al.,BioTechniques, 1996,21, 625–626) does not interfere with further MDPF staining and fluorescent detection of these bands transferred to PVDF membranes. Thus, Nile red and MDPF staining can be performed sequentially, allowing the rapid monitoring of total protein patterns on both the electrophoretic gel and Western blot. Using the conditions described in this study, MDPF staining does not preclude furtherN‐terminal microsequencing and immunodetection of specific bands with

ISSN:0173-0835    

DOI:10.1002/elps.1150191408

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractWe have developed a new mixed‐dye protein staining method that is simple, rapid, and sensitive. A freshly prepared mixture of calconcarboxylic acid (NN, 0.02%) and rhodamine B (RB, 0.04%) in 40% methanol / 7% acetic acid, was used as a staining solution. RB acts as an auxiliary agent to inhibit the binding of NN to the gel matrix, reducing the background staining and therefore enhancing the protein staining by NN. This mixed‐dye staining method reduces the total staining and destaining time to less than an hour, and increases the sensitivity to 25 ng of bovine serum albumin, which is greater than the 100 ng sensitivity limit of Coomassie Brilliant Blue R‐250 (CBBR) sta

ISSN:0173-0835    

DOI:10.1002/elps.1150191409

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractA highly sensitive method for detecting deoxyribonucleases (DNases) I and II on an electrophoresed gel is described. A dried agarose film sheet containing DNA as a substrate and a buffer reagent was placed in contact with the gel surface after electrophoresis (DAFO method, Yasudaet al.,Anal. Biochem.1989,183, 84–88). After an appropriate incubation period, the film sheet was peeled off and stained with SYBR‐Green I (SG), and then the DNase isozyme bands were detected using a fluorescence image analyzer. We could detect pg levels of the DNases (DNase I, 2 pg; DNase II, 2pg), which represents a 32‐ to 128‐fold increase in sensitivity compared with the original DAFO method using ethidium bromide (EB) as the fluorescent dye. A combination of this new detection method and isoelectric focusing electrophoresis in polyacrylamide gel allowed accurate DNase I typing from 1 μL human serum. This new technique has been named SG‐DAFO, after its original dried agarose film overlay method using EB

ISSN:0173-0835    

DOI:10.1002/elps.1150191410

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractThe antibody response development during polyclonal antibody production is a relevant parameter to monitor during the immunization period to be able to optimize the immunization protocol and to determine the optimal antibody harvest time. Although rabbits and other mammals are most often used for polyclonal antibody production, the chicken is a relevant alternative. There are both scientific reasons, economic reasons, and animal welfare reasons to consider when choosing the chicken instead of a mammal for this purpose, because antibodies in generous quantities can be harvested from the egg yolk. This study compared different assays for measuring antibody response in rabbit and chicken serum. An inhibition liquid phase absorption assay (ILPAA), a rocket immunoelectrophoresis (RIE) assay, and a line immunoelectrophoresis (LIE) assay were compared to ELISAs. The ELISA proved to be the most useful assay for routine use, as it was less time‐consuming and because the assay could easily be adapted to both serum antibody types. However, electrophoretic assays were the most useful as combined analytical and quantitative tools and must be considered essential when analyzing specificities of polyclonal antibody preparation

ISSN:0173-0835    

DOI:10.1002/elps.1150191411

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY