Applications of consensus polymerase chain reaction with subsequent electrophoretic distinction of amplificates.
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.
Krause
G
G
Medizinische Klinik, Klinikum Innenstadt der Universität, München, Germany. 101356.242@compuserve.com
Oduncu
F
F
Kaltstein
A
A
Pusl
T
T
Pachmann
K
K
Gerzer
R
R
D000818:Animals; D016384:Consensus Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D015322:Gene Rearrangement, B-Lymphocyte; D006162:Guanylate Cyclase / Q000096:biosynthesis / Q000235:genetics; D006168:Guinea Pigs; D006801:Humans; D007147:Immunoglobulin Light Chains / Q000032:analysis; D009696:Nucleic Acids / Q000032:analysis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D011971:Receptors, Immunologic / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000032:analysis; D015183:Restriction Mapping
D017931:DNA Primers; D007147:Immunoglobulin Light Chains; D009696:Nucleic Acids; D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins; D006162:Guanylate Cyclase