The Assessment of Detection of Quantitative Alteration in Microsatellite DNA by Real-Time PCR Technology

• Main Authors: Jia-Yu Kang, 康佳瑜
• Other Authors: Yi-Chih Chien
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/20576491999785400516

碩士 === 國立彰化師範大學 === 生物學系 === 95 === Abstract Genetic alteration plays an important role in cancer progression. Loss of heterozygousity (LOH), caused by allelic imbalance, is one of the distinctive genetic alteration mechanisms in the process of cancer progression. The way Gene Scan detects LOH is limited to the ratio of heterozygosity, while QuMA not only solves the problem, but it also differentiates whether a genetic alteration gains or loses. The following experiment is designed, by means of QuMA, to establish a system which can detect the genetic alteration, and to compare if the results correspond to those detected by Gene scan, in an attempt to substitute for Gene Scan. Used by Gene scan, the first rank of the markers, D17S849 (17p), D8S505 (8q), and the second rank of the marker, D16S3091 (16q), are adopted to perform relative quantification of tissue, plasma, and blood in liver cancer in the experiment. To the internal controls, β-actin, and the original density of DNA gained by Fluorometry are used for revision. The results are as follows: (1) in the experiment on tissue using β-actin as an internal control, 82.1% of results showed by D17S, 90％ by D8S, and 75％ by D16S accord with those by Gene Scan, revealing high correspondences based on Kappa coefficient of agreement（p＝0.01、p＝1.6×10-4、p＝0.02）.When the original density of DNA gained by Fluorometry is adopted as an internal control, the corresponding results are 60.7％ by D17S, 53.3％ by D8S, and 66.7％ by D16S. (2) In the experiment on plasma using β-actin as an internal control, the consistent results are 33.3％ conducted by D17S, 65.2％ by D8S, and 46.7％ by D16S, while the equivalent results in using the original density of DNA gained by Fluorometry as an internal control are changed to 35％ by D17S, 27.3％ by D8S and 66.7％ by D16S. The experiment indicates that β-actin used by QuMA as an internal control shows more accuracy than Fluorometry. The reason why Fluorometry is inappropriate might be that Fluorometry did not undergo the process of PCR reaction. The results in plasma by QuMA differ greatly from those by Gene Scan. The cause might be that Gene Scan was not sensitive to detect microsatellite DNA of plasma, because many samples were not detected by it, and the period of half-life of plasma was short, leading to low ratio of consistency. (3) In the experiment on blood, according to QuMA, the relative amount of people with lung cancer and that of people without the cancer are verified by discriminate analysis. The ratio of accuracy in discriminating the two classes of people amounts to 91.9% based on discriminant analysis. Therefore, whether people have lung cancer or not can be differentiated by the relative amount. In further analysis of the three markers used by QuMA and Gene scan, there is no obvious relationship between the overall survival and disease free survival to the markers. The reason is that Gene scan adopted the three markers according to the ratio of LOH in tissue and plasma, exclusive of the assessment of prognosis. Also, the experiment found out that the original density of DNA gained by Fluorometry is manifestly related to metastasis（p=0.0048）. The experiment verifies that QuMA is in fact not restrained to the ratio of heterozygosity, and is fast to screen. In addition, QuMA is more convenient for manipulation than Gene Scan. It is hoped that QuMA will be applied to detect cancer in its early stage and for prognosis in the future.