Study On Using The Real Time Polymerase Chain Reaction To Quantitate BCR-ABL MRNA

The total number of tumor cell of acute leukemia before chemotherapy is about1010~1012, even when the patient has got complete remission after chemotherapy,the number of tumor cell only decrease to about 108~109, that is about1/1000~10000 of the total number of the cell in bone marrow, further more, thereare a little tumor cell exist in other tissues, which is called as minimal residualdisease (MRD), and it is the base of relapse. In order to cure leukemia and let thepatient get long-time survival without tumor, we should not only find there isMRD or not, but also to quantitate the MRD, because our immune system can getrid of a certain number tumor cell, at least the immune system can control itsproliferation. That is, if there is no quantitative value of MRD, let alone detectingMRD is useful to clinical instruction.People have done a lot of work to obtain the method to diagnose leukemia. Atthe early stage, there were mainly routine bone-marrow morphology andchromosome band forms analysis.During 1980s~1990s, with the development of molecular biology and theoccurrence of flow cytomatric cell sorting (FACS), we can diagnose leukemiawith FACS and fluorescence in situ hybridization(FISH), which isn't enough todiagnose the tumor cells. FACS can find several tumor cells, but it is still notenough to diagnose MRD, too.PCR at its early stage was semi-quantitative methods. The semi-quantitativereverse-transcription polymerase chain reaction (RT-PCR) results are typicallygenerated by frequent sampling of the RT-PCR reaction mixture followed bydot-blot analysis, this doesn't lead itself to easy automation and can increase thelikelihood of contamination and of detection of false positives. 3The application of fluorescence techniques to the RT-PCR, together withsuitable instrument capable of combining amplification, detection andquantification, had led to the development of kinetic RT-PCR methodologies thatare revolutionsing the possibilities for quantitating nucleic acid. It significantlysimplifies the process of producing reproducible quantification of nucleic acid andpromises to overcome the limitations suffered from the problems inherent in PCR.There are several techniques that can be used to detect amplified product. Theyuse fluorescent dyes and combine the processes of amplification and detection ofan nucleic acid target to permit the monitoring of PCR reactions in real-timeduring the PCR.The simplest method uses fluorescent dyes that bind specificallyto double-strand DNA, the others rely on the hybridization of the fluorescent-labeled probes to the correct amplicon, these methods include: molecular beacons,TaqMan probes, and so on. Their sensitivity eliminates the need for asecond-round amplification, and decrease opportunities for generatingfalse-positive result. These methods differ in their specificity, although at lateramplification cycles all can show artefacts that do not correlate to specific productaccumulation, and most of them depend on successful hybridization of the probewith the target DNA, obviating the need for post-PCR southern analysis, orsequencing to confirm the identity of the amplicon.The fluorescence values are recorded during every cycle and represent theamount of product amplified to that point in the amplification reaction, the moretemplate present at the beginning of the reaction, the fewer number of cycles ittake to reach a point in which the fluorescent signal is first recorded as statisticallysignificant above back-ground, this point is defined as the Ct, and will alwaysoccur during the exponential phase of amplification.Thanks to the linear response of Ct value and the logarithm of the initial copynumber of the template, according to the standard dilutions lf the recombinedplasmid and their Ct values, we can get a standard curve by plotting the Ct valueagainst the logarithm of the initial copy numbers. Then the copy number ofexperimental RNAs can be calculated after real-time amplification from the linerregression of that standard curve.We take the method of hydrolysis probe—Taq Man assays, it utilizes the5'-nuclease activity of the DNA polymerase to hydrolyze a hybridization probebound to its target amplicon. The probe contains a fluorescent reporter dye at its 5'end, the emission spectrum of which is quenched by a second fluorescentquenched dye at its 3'end. After denaturation, primers and probe anneal with thetemplate, the quencher at 3'end quenches the signal from the reporter at 5'end.When the strand displacement occurring, the polymerase displaces and hydro-lyses the labeled probe, so the fluorescent dye is released from the probe, and thefluorescence is detected.Our subject is BCR-ABL oncogene, which is the result of translocation oft(9;22), it conform the Ph's chromosome which can occur above90% of chronicmyelocytic leukemia.Through our test, we have established a real-time PCR method to detectBCR-ABL oncogene transcriptions, and we have got the standard curve, thecoefficient of correlation(R) is 0.98; the sensitivity is 10 recombined plasmids, thecoefficient variation(CV) of repetition and stability is 2.19% and 3.21%respectively. So we get conclusions as follow: 1.The real-time PCR has perfectsensitivity, repetition, and specificity; 2.It can quantitate the copy number of...