Time-resolved immunofluorometric determination of specific mRNA sequences amplified by the polymerase chain reaction.
We report a highly sensitive time-resolved immunofluorometric method for quantification of polymerase chain reaction (PCR)-amplified mRNA sequences. The PCR primers are labeled at their 5' ends, one with biotin and the other with a hapten. The modified primers are incorporated, during PCR, in the amplified product. The PCR product is captured, through its biotin moiety, to a streptavidin-coated solid phase and subsequently is detected with an alkaline phosphatase-labeled antibody. The phosphate ester of fluorosalicylic acid is used as a substrate. The fluorosalicylate produced forms a highly fluorescent ternary complex with Tb(3+)-EDTA, which is measured by time-resolved fluorometry. We chose the determination of PCR-amplified chronic myelogenous leukemia-specific mRNA as a model system. mRNA molecules corresponding to 0.1 leukemic cell in the presence of 0.5 million normal cells may be detected (signal-to-background ratio of 1.5). The method provides a sensitive and rapid nonisotopic alternative to Southern blot and hybridization with radioactive probes.
Bortolin
S
S
Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada.
Christopoulos
T K
TK
D000469:Alkaline Phosphatase / Q000737:chemistry; D000906:Antibodies / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry; D001483:Base Sequence; D001710:Biotin / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004076:Digoxigenin / Q000737:chemistry; D004492:Edetic Acid / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D015200:Fluoroimmunoassay; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis* / Q000737:chemistry; D019809:Streptavidin; D013379:Substrate Specificity
D000906:Antibodies; D001426:Bacterial Proteins; D017931:DNA Primers; D012333:RNA, Messenger; D001710:Biotin; D019809:Streptavidin; D004492:Edetic Acid; D000469:Alkaline Phosphatase; D004076:Digoxigenin