[Real time quantitative PCR].
So far, quantitative techniques, such as PCR and FISH, have been used to detect of DNA and RNA. However, it is difficult to measure and compare the exact amount of amplified products with the results of endpoint analysis in conventional PCR techniques. Theoretically, there is a quantitative relationship between amount of starting target sequence and amount of PCR product at any given cycle. The development of real-time quantitative PCR (RQ-PCR) has eliminated the variability associated with conventional quantitative PCR, thus allowing the routine and reliable quantitation of PCR products. Detection of fluorescence during the thermal cycling process can be performed using iCycler(Bio-Rad), the GeneAmp 5700 or 7700(ABI-PRISM), and Light-Cycler(Roche). Two fluorogenic probes are available for use on real time quantitation. The fluorogenic 5'-nuclease assay(Taqman method) uses a fluorogenic probe to enable the detection of a sequence specific PCR product. Fluorogenic probe is incorporated with the reporter dye on the 5' end and the quencher on the 3' end. The second method uses SYBR Green I dye which is a highly specific double-stranded DNA binding dye. Real-time PCR is able to be possible exact quantitation of DNA and RNA much more precise and reproducible because it is based on CT values acquired during the exponential phase of PCR rather than endpoint. In this review, the detail protocol of real time quantitative PCR technique will be introduced and our recently developed system for exact quantitation of BCR-ABL fusion gene in CML is going to be described.
Kim
D W
DW
Molecular Hematology Laboratory, Catholic Hemopoietic Stem Cell Transplantation Center, The Catholic University of Korea, Seoul.
D001483:Base Sequence; D017931:DNA Primers; D015342:DNA Probes; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D016044:Fusion Proteins, bcr-abl / Q000032:analysis; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D017931:DNA Primers; D015342:DNA Probes; D005456:Fluorescent Dyes; D016044:Fusion Proteins, bcr-abl