The Construction Standard Contours Of The Tumour Metastasis Related Gene Tiam1 MRNA TaqMan Real-time Fluorescence Quantitive PCR

The metastasis is one of the biologic characteristics of cancer andit's the main cause of death of cancer patients.It's gene expression andregulation, molecular biology behavior and the relevance ofrecurrence and prognosis isn't be known clearly . So the research oftumour's metastasis become a current major focus . Tumor'smetastasis is a complex process involving a series of genetic changes,including tumors break off from primarily site , infiltrating it'ssurrounding organizations, acrossing the vessel wall into thecirculatory system, escaping from the immune system's attacks inthe circulatory system, finally throughing vessel and adhere toendothelial cell, inducing the form of blood vessels and lymphvessel, proliferating to form metastasis in remote parts. This wholeprocess is regulated by many genes. Many findings suggest that Tlymphoma invasion and metastasis inducing factor 1 (Tiam1) isexpressed in almost all human and Rodentia zoic tumor cell lines. Itsincreased expression is related to species of tumor cell's invasion andmetastasis. So Tiam1gene was called the tumour metastasis relatedgene. Tiam1 is Rac1 the specific guanine nucleotide exchange factor(GEF). Stimulated by extracellular signal, Tiam1 can be to promotefrom the inactive Rac1-GDP to the active Rac1-GTP. The activeRac1-GTP participates reconstruction of cystoskeleton,cell adhesion,cell movement, cell proliferation and differentiation, apoptosis and soon. So Tiam1 participated the tumour's invasion and metastasis.In the past, the research to Tiam1 gene was on the qualitation orsemi-quantitative level, which was limited on explaining somequestion's essence and ultraminim detecting trace copy. So the use ofa more accurate and more sensitive detection methods will become ahot topic of genetic research. In 1996, the Applied Biosystemscompany first introduced real-time fluorescence quantitative PCR(real-time FQ-PCR) technology. Real-time FQ-PCR is a method thatadding fluorophore to PCR reaction system,using fluorescence signalaccumulation to real time monitoring the whole PCR proceeding,finally through standard curve to quantitive analysis unknowntemplate. The technology makes PCR leaping form qualitation toquantitation.The experiment adopt the TaqMan probe technology. It added aspecific fluorescence probe to PCR amplification system. The probecan hybrid specifically to DNA template.The 5'end of probe wassigned a fluorescence reporter dye and its3'end was signed afluorescence quencher dye. When probe was integrated, thefluorescence sign was quenched by fluorescence quencher sign at 3'end. In anneal stage, probe and primer hybrid with template specificly.In elongation stage, the pclymerase hydrolyzes and displaces thelabeled probe, and the quench effect was removed, then thefluorescence signals released from the prome andis detected. Whenamplificated a DNAchain, there is a fluorescence molecular formation. It implemented the synchronization of the cumulation offluorescence signal and PCR product .So that technology canaccurately fix quantify templates.The experiment showed that the Tiam1cDNA was clonedsuccessfully and has gained stabile recombinant plasmid. We usedTaqman probe technology to amplify the standard preparationof100-107/ul reorganization plasmids. The statistical analysis showedthat there is a good linear relationship between Ct values andlogarithm of recombinant plasmid standard preparation'sconcentrations, and Tiam1mRNA quantitative real-time fluorescencePCR standard curve was constructed successfully. We founded amethod of quantitating Tiam1 mRNAwith Taqman fluorescence probe,which established foundation for accurate quantitative testing of thevarious organizations Tiam1 mRNA level. So we get conclusion asfollow: 1. utilized TaqMan technology, we constructed successfullyrecombinant plasmid standard preparation of tumour metastasisrelated gene Tiam1 mRNA for real-time fluorescence quantitativePCR. 2. The real-time fluorescence quantitative PCR appearancedrawed standards curve.Ct value and the copy number of primitivetemplates had a good linear relationship. the statistics authenticatedthat the methodology was stability and repetitive. 3. Compared withordinary PCR, real-time fluorescence quantitative PCR technology arenot only quantifiable, but also can detect genetic copy which can'tbe done by ordinary PCR, So it can be used for the detection of moreminor ailments.