TIME-RESOLVED IMMUNOFLUOROMETRIC DETERMINATION OF SPECIFIC MESSENGER-RNA SEQUENCES AMPLIFIED BY THE POLYMERASE CHAIN-REACTION

被引：16

作者：

BORTOLIN, S ^{[1
]}

CHRISTOPOULOS, TK ^{[1
]}

机构：

[1] UNIV WINDSOR,DEPT CHEM & BIOCHEM,WINDSOR N9B 3P4,ON,CANADA

来源：

ANALYTICAL CHEMISTRY | 1994年 / 66卷 / 23期

关键词：

D O I：

10.1021/ac00095a029

中图分类号：

O65 [分析化学];

学科分类号：

070302 ; 081704 ;

摘要：

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 Tb3+-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.

引用

页码：4302 / 4307

页数：6

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