COX-2基因啟動區突變點檢測以及快速篩檢的方法之研究

Abstract

隨著人類基因體計畫的進展，人們發現個體基因的多型性有助於解釋個體的表型差異、不同群體和個體對疾病，特別是對複雜疾病的易感性、以及對各種藥物的耐受性和對環境因素的反應。但因我們所能取得的樣本有限，因此以微量的樣本所萃取出DNA來做各種鑑定及研究，有其重要性。 我們研究的重點就是簡單、容易地把微量DNA萃取出來，並利用些特殊方法，將所萃取的微量DNA大量複製出我們想要的片段來，又因有相關證據表示COX-2的表現與癌症具有關聯。所以我們藉著最新的分析技術，試著從大量樣品中，來篩選出可能有COX-2基因變異的個體。 本論文可分三部分，首先我們利用牙科紙針從10位台大醫院病人的口腔中吸取唾液及洗牙後於牙齦溝滲出之血液，所取之紙針利用QIAamp DNA Micro kit 抽取出其中之DNA，並以IL-1β基因引子進行聚合酶連鎖反應（PCR），最後再以洋菜膠（agarose）電泳顯示出結果。 第二部分，樣品取自台大醫院牙科就診的201位病人，其中187位是沒得過口腔癌的人，14位為現在口腔癌的病人或有口腔癌病史的人，以萃取之DNA來複製長片段的COX-2基因啟動區，使用更精密的巢狀聚合酶連鎖反應（nest PCR），並測試此方法複製長片段基因之成功率。最後，利用最新的high-resolution儀器來分析篩選出COX-2基因啟動區可能有變異的樣品。 結果顯示，所有的唾液及血液之微量DNA皆能由QIAamp的方式來萃取出來，並且IL-1β基因（249 b.p）經過PCR的放大後，都可以在電泳膠上清楚的顯示。在使用nested PCR，來複製長片段COX-2基因啟動區方面有95%以上的成功率。而且我們用high-resolution儀器分析也能迅速地將全部的樣品基因分出兩種波形，並藉由這兩種波形的差異來篩選出可能有變異的樣品，進而以核酸定序來證實之。 往後我們可用紙針蒐集血液和唾液方式來替代較具侵入性之檢驗法，這不但有容易操作、容易取得、不佔空間、節省成本等優點，並且可以降低受檢者之恐懼感及不適，大大地提升其接受檢驗之意願。nested PCR可以有95%以上的成功率將長片段基因複製出來，如此可以用非常微小的量來大大地增加DNA複製的效率。利用最新的high-resolution儀器分析，將來我們不需花時間將所有的樣品定序，而且不需花很多金錢來做real-time PCR分析，只需要一組primer，一點LC Green dye和一台PCR儀器及HR-1儀器，就可以簡單、不貴且迅速地分析出DNA的基因型和變異型。 High-resolution儀器分析將會是未來檢測DNA變異的主流方法之一。

Background Data: Since the marked advance of Human Genome Project, analysis of genetic polymorphism has been popularly used to explain the individual difference in susceptibility to various diseases, such as infection of complicated diseases, sensitivity of medication and environment. However, analysis of gene polymorphisms is sometimes not feasible due to limited DNA available. Objective: A simple and easy method to extract DNA from minimal amount of saliva and blood for PCR amplification of gene is necessary. Some studies indicated the relation between COX-2 expression and cancer. Thus, we try to design an analytical technique to compare the COX-2 promoter region polymorphism in clinical healthy and oral cancer patients. Materials and Methods: This study divided three parts. First, we collected saliva and blood with paper points after full mouth scaling in NTUH. The tips of paper points were used to extract DNA using QIAamp DNA Micro kit. PCR was used to amplified IL-1βgene from isolated DNA, subjected to agarose gel electrophoresis, stained with ethidium bromide. Second, we followed the same method to collect samples and the samples were 201 patients in NTUH, including 187 healthy control subjects and 14 patients who had oral cancer or medical history of oral caner. DNA was isolated and used to amplified the promoter region of COX-2 gene by more sensitive method “nested PCR”. Finally, we utilized the latest analytical technique “high-resolution melting analysis” and nucleotide sequencing to screen some doubtful samples with mutation of COX-2 promoter region. Results: We successfully extracted DNA from all blood and saliva samples by QIAamp DNA isolation kits. After PCR amplification of IL-1β genes (249 b.p), the specific DNA bands were clearly identified on agarose gels after ethidium bromide staining. Moreover, by using Nested PCR, the successful rates to amplify long genetic section of COX-2 promoter region were above 95%. We further utilized high-resolution melting analysis to detect two patterns of the melting curves. According to these two types of curves, we could screen doubtful samples and make further confirmation of gene polymorphism sites by DNA nucleotide sequencing.

Conclusion: The results indicate that we can use paper point to substitute the invasive methods for collecting blood and saliva for DNA analysis in the future. The advantage is easy to operate and obtain, not to occupy the space, and the economical cost. Futhermore, people can reduce fear and receive examination by this method. We can amplify long genetic section above 95% by nested PCR which can greatly increase efficiency to amplify tiny amount of DNA. Utilizing high-resolution melting analysis, we can not spend time to sequence all samples, and can not spend money to do real-time PCR. SNP genotyping will be simple, rapid, and inexpensive, requiring only one pair of primers, a little LC Green dye, one PCR machine, and one HR-1 machine. High-resolution melting analysis will be the best methods of SNP genotyping in the future.