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  • 1
    Electronic Resource
    Electronic Resource
    Applications of consensus polymerase chain reaction with subsequent electrophoretic distinction of amplificates (1997)
    Weinheim : Wiley-Blackwell
    Electrophoresis 18 (1997), S. 1098-1102 
    Details
    ISSN: 0173-0835
    Keywords: Polymerase chain reaction ; Single-strand conformation polymorphism ; Gene expression ; Guanylyl cyclases ; Leukemia ; Antigen receptor rearrangements ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Conserved sequences within gene families permit the design of consensus primers that match several members of a given class of homologous genes. Polymerase chain reaction (PCR) products obtained with such consensus primers were characterized by restriction mapping or single-strand conformation polymorphism (SSCP) analysis, using precast polyacrylamide minigels and automated silver staining. Examples for the electrophoretic distinction of consensus amplificates are presented in the fields of guanylyl cyclase expression studies and in the determination of B-cell clonality in human blood samples. Guanylyl cyclase expression in inner ear tissues of guinea pigs was investigated by reverse transcription PCR using consensus primers with specificity for the subclass of particulate guanylyl cyclases. The resulting PCR products were assigned to three representatives of this group by restriction mapping. The consensus PCR approach enabled the detection of an unexpected receptor type, namely guanylyl cyclase C, in the inner ear. The distinction by SSCP analysis of denatured consensus amplificates was appropriate for the identification of clone-specifically rearranged immunoglobulin heavy chain genes of B-lymphocytes. Genomic DNA isolated from blood samples of leukemia patients served as the template for the consensus amplification of clone-specific VDJ rearrangements. Rapid distinction and re-identification of consensus PCR products was achieved by SSCP analysis for regular antigen receptor rearrangements and for t(14; 18) translocations. The potential of these procedures for detecting leukemia or lymphoma clones when monitoring minimal residual disease was assessed.Presented at the “Elektrophorese Forum '96” meeting of the German Electrophoresis Society, held at the Technical University Munich, October 23-25, 1996.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150180712
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  • 2
    Electronic Resource
    Electronic Resource
    Distinction of weakly homologous cDNA amplificates by single-strand conformation polymorphism analysis: Application to guanylyl cyclase isozymes (1994)
    Weinheim : Wiley-Blackwell
    Electrophoresis 15 (1994), S. 557-561 
    Details
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: By use of the polymerase chain reaction (PCR), uniform amplification products of 225 to 240 bp length were obtained from five cDNA clones representing different types of guanylyl cyclases. These short DNA double strands were differentiated by single-strand conformation polymorphism (SSCP), using polyacrylamide gel electrophoresis with the Pharmacia Phast-System. Following heat denaturation, the samples were separated on native polyacrylamide gels at different running temperatures. Nucleic acids on the gel were detected by an automated silver stain procedure. Using 7.5% homogeneous or 4-15% gradient polyacrylamide gels at a temperature of 12°C, single-strand conformations of amplificates, representing three different particulate guanylyl cyclases and the two subunits of soluble guanylyl cyclase, were differentiated. The characteristic banding patterns resulting from dissimilar migration of the single-strand conformations were assigned to different guanylyl cyclase types. For the enzyme family of guanylyl cyclases, the feasibility of a combined PCR and electrophoresis approach for analyzing the expression of related genes was demonstrated. This application of the PCR-SSCP technique provided a rapid and sensitive tool for the characterization of PCR products obtained with a common primer pair and suggested its use for investigating the tissue distribution of gene expression within a class of homologous proteins.
    Additional Material: 1 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150150175
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