title: Electrochemical branched-DNA assay for polymerase chain reaction-free detection and quantification of oncogenes in messenger RNA.
abstract: We describe a novel electrochemical branched-DNA (bDNA) assay for polymerase chain reaction (PCR)-free detection and quantification of p185 BCR-ABL leukemia fusion transcripts in the population of messenger ribonucleic acid (mRNA) extracted from cell lines. The bDNA amplifier carrying high loading of alkaline phosphatase (ALP) tracers was used to amplify the target signal. The targets were captured on microplate well surfaces through cooperative sandwich hybridization prior to the labeling of bDNA. The activity of captured ALP was monitored by square-wave voltammetric (SWV) analysis of the electroactive enzymatic product in the presence of 1-naphthyl phosphate. The voltammetric characteristics of substrate and enzymatic product as well as the parameters of SWV analysis were systematically optimized. A detection limit of 1 fM (1 x 10(-19) mol of target transcripts in 100 microL) and a 3-order-wide dynamic range of target concentration were achieved by the electrochemical bDNA assay. Such limit corresponded to approximately 17 fg of the p185 BCR-ABL fusion transcripts. The specificity and sensitivity of assay enabled direct detection of target transcripts in as little as 4.6 ng of mRNA population without PCR amplification. In combination with the use of a well-quantified standard, the electrochemical bDNA assay was capable of direct use for a PCR-free quantitative analysis of target transcripts in mRNA population. A mean transcript copy number of 62,900/ng of mRNA was determined, which was at least 50-fold higher than that of real-time quantitative PCR (qPCR). The finding was consistent with the underestimation of targets by qPCR reported earlier. In addition, the unique design based on bDNA technology increases the assay specificity as only the p185 BCR-ABL fusion transcripts will respond to the detection. The approach thus provides a simple, sensitive, accurate, and quantitative tool alternative to the qPCR for early disease diagnosis.
authors.affiliation: Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
authors.forename: Ai-Cheng
authors.initials: AC
authors.lastname: Lee
authors.forename: Ziyu
authors.initials: Z
authors.lastname: Dai
authors.forename: Baowei
authors.initials: B
authors.lastname: Chen
authors.forename: Hong
authors.initials: H
authors.lastname: Wu
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.forename: Aiguo
authors.initials: A
authors.lastname: Zhang
authors.forename: Lurong
authors.initials: L
authors.lastname: Zhang
authors.forename: Tit-Meng
authors.initials: TM
authors.lastname: Lim
authors.forename: Yuehe
authors.initials: Y
authors.lastname: Lin
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism; D021121:Branched DNA Signal Amplification Assay; D004563:Electrochemistry; D016044:Fusion Proteins, bcr-abl / Q000235:genetics*; D006801:Humans; D015466:Leukemia, Myeloid, Chronic-Phase / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
chemical_list: D012333:RNA, Messenger; D016044:Fusion Proteins, bcr-abl; D000469:Alkaline Phosphatase