臺灣博碩士論文加值系統

固定核苷酸於固相載體，廣泛應用在分子生物學和臨床醫學上，包括親和性層析、胜肽與低聚核苷酸的合成、生物感測器以及基因晶片等。本研究主要是利用分散聚合法，將甲基丙烯酸環氧丙酯和苯乙烯兩種單體共聚合得到大小約2微米的非磁性高分子微米顆粒，利用化學改質方式，將顆粒表面官能基改質成胺基與醛基，分別利用共價鍵結與非共價鍵結方式固定單股低聚核苷酸，再進行雜合試驗。同時考量磁性顆粒在應用上有容易分離回收的優點，因此將磁性物質包埋入上述高分子微粒，形成具有磁性的顆粒，以相同的方法固定單股低聚核苷酸之後，進行雜合效率之比較。
本研究固定單股低聚核苷酸為探針進行雜合，發現雜合時間以20分鐘至1小時為宜。而增加探針濃度則固定在顆粒上的探針鍵結量越高，標的低聚核苷酸的雜合量也越高，經十次反覆雜合試驗，結果顯示多次雜合不會影響其雜合效率，亦不受冷凍處理之影響。同時證實高分子微米顆粒固定探針之雜合具有專一性，因此具有極高之應用價值。綜合各種顆粒固定單股低聚核苷酸之鍵結量與雜合效率，固定方式以「顆粒表面鍵結5’ amino primer2」最優，「顆粒表面鍵結5’ biotin primer2」次之，「顆粒表面鍵結5’p primer2」最差，而顆粒種類磁性又優於非磁性，惟磁性顆粒5’端磷酸固定單股低聚核苷酸的雜合系統，顆粒在改質過程中會流失部分磁性物質，在應用上是一項缺失。

Immobilized oligonucleotides have been widely used in the studies of molecular biology and clinical analysis, including affinity chromatography, peptide and oligonucleotides synthesis, biosensor and DNA-microchip technology. In this study, non-magnetic polymer microbeads having a particle diameter of ca. 2.0 µm were prepared by the dispersion polymerization of glycidyl-methacrylate(GMA) and styrene. For the application in DNA hybridization, the copolymerized beads were chemically modified to introduce aldehyde and amino groups, which were allowed for covalent or non-covalent linking of single-stranded oligouncleotides. At the same time, magnetic polymer microbeads, taking the advantages of easy separation in particle use, were also prepared by entrapping of magnetic material in the polymer microbeads and employed for the immobilization of single-stranded oligonucleotides. The efficiencies of hybridization through the use of magnetic and non-magnetic microbeads were compared.

In the present study, a specific sequence of immobilized single-stranded oligouncleotides was assigned as the probe for the hybridization. The most appropriate time for hybridization was determined to be in the range from 20 minutes to one hour. Moreover, increasing the incubated concentration of probe increased the amount of probe immobilized on the microbeads. As the amount of bound probe increased, the amount of hybridization of the target became greater. According to the results from ten-times repeated hybridizations, the efficiency of hybridization won’t be significantly affected neither by the repetitive runs of hybridization nor the freezing process. In the meanwhile, it was proved that the hybridization of fluorescent target DNA to the immobilized probe on the polymer microbeads was unique, suggesting an extremely high value of application. Analyzing the efficiency of hybridization from different kinds of microbeads coupled with single-stranded oligouncleotides, it was found that the best way to prepare immobilized probe was the binding of 5’ amino-containing oligonucleotide on microbeads. The immobilization of 5’ biotin-labeled oligonucleotide on the microbeads bound with avidin was the second choice. The inferior one was the binding of 5’ phosphate-containing oligonucleotide on microbeads. In all oligonucleotide-immobilized protocols, magnetic microbeads were always better than non-magnetic ones. However, during the surface modification process for the binding of 5’ phosphate-containing oligonucleotide on microbeads, magnetic material could be lost to some extant, which may be a defect in application.

中文摘要……………………………………………………………………I
英文摘要…………………………………………………………………III
目錄…………………………………………………………………………V
圖目錄………………………………………………………………………X
表目錄 ……………………………………………………………………XI
第一章 緒論…………………………………………………………………1
1.1研究動機與目的 ………………………………………………1
1.2文獻回顧 ………………………………………………………3
1.2.1固定DNA ……………………………………………… 3
1.2.2親和性吸附固定DNA ……………………………… 4
1.2.3化學反應共價鍵結DNA ……………………………… 5
1.2.4雜合最適溫度……………………………………………6
1.2.5雜合後之變性方法………………………………………7
1.2.6包埋磁性顆粒的高分子顆粒技術………………………8
1.2.7固定單股低聚核苷酸在雜合上的應用…………………9
第二章實驗藥品與設備 ………………………………………………… 11
2.1實驗藥品 …………………………………………………… 11
2.1.1高分子顆粒製備 ………………………………………11
2.1.2顆粒表面之乙二胺改質 ………………………………12
2.1.3顆粒表面之戊二醛改質 ………………………………12
2.1.4 PS/GMA顆粒上醛基含量的測定 ……………………12
2.1.5固定卵白素與接合5’-biotin primer2 …………………13
2.1.6顆粒表面之二級胺改質 ………………………………13
2.1.7固定5’P primer2 ………………………………………14
2.1.8固定5’amino primer2 …………………………………14
2.1.9雜合…………………………………………………15
2.2實驗設備 ………………………………………………………16
第三章 實驗方法與步驟 …………………………………………………18
3.1非磁性顆粒改質為二級胺 ……………………………………18
3.1.1共聚合高分子顆粒之製備……………………………18
3.1.2非磁性顆粒表面環氧基之胺基改質…………………19
3.1.3利用非磁性顆粒表面胺基固定5’P primer2…………21
3.1.4定量固定在非磁性顆粒上之5’P primer2的量………21
3.1.5製作anti-primer2檢量線 ……………………………23
3.1.6非磁性顆粒改質為胺基之雜合……………………25
3.2非磁性顆粒表面改質為醛基…………………………………26
3.2.1共聚合高分子顆粒之製備……………………………26
3.2.2非磁性顆粒表面之乙二胺改質………………………27
3.2.3具胺基之非磁性顆粒表面進行戊二醛修飾…………27
3.2.4非磁性顆粒表面醛基含量的測定……………………28
3.2.5非磁性顆粒改質為醛基鍵結卵白素固定
5’biotin primer2 ……………………………………… 30
3.2.6非磁性顆粒改質為醛基鍵結卵白素固定探針之雜合33
3.2.7非磁性顆粒表面改質為醛基固定 5’amino primer2… 34
3.2.8非磁性顆粒改質為醛基固定含5’胺基探針之雜合…34
3.3磁性顆粒……………………………………………………36
3.3.1製備磁性顆粒……………………………………… 36
3.3.2磁性顆粒表面官能基之改質……………………… 36
3.3.3磁性顆粒改質後之雜合 ……………………………… 36
第四章實驗結果與討論………………………………………………… 37
4.1甲基丙烯酸環氧丙酯與苯乙烯共聚物顆粒之製備……… 37
4.2雜合時間之探討…………………………………………… 38
4.3相同雜合時間下不同起始標的濃度雜合效率之探討…… 40
4.4非磁性顆粒官能基改質為二級胺固定探針 ……………… 42
4.4.1 非磁性顆粒表面胺基之改質 ……………………… 42
4.4.2 非磁性顆粒以胺基共價固定探針(5’P primer2)
之定量 …………………………………………… 43
4.4.3 非磁性顆粒改質為二級胺鍵結探針之
多次雜合之效率 …………………………………… 43
4.5 非磁性顆粒官能基改質為醛基固定探針 ………………… 46
4.5.1非磁性顆粒表面醛基之改質 ………………………… 46
4.5.2非磁性顆粒表面醛基之定量 ………………………… 46
4.5.3 固定在非磁性顆粒上卵白素之定量 ……………… 47
4.5.4非磁性顆粒改質為醛基固定探針之雜合效率 ……… 48
4.6磁性顆粒表面官能基改質為二級胺固定探針 ……………… 50
4.6.1磁性顆粒表面胺基改質 ……………………………… 50
4.6.2磁性顆粒以胺基共價固定探針(5’P primer2)之定量50
4.6.3磁性顆粒改質為二級胺固定探針之雜合效率 ……… 51
4.7磁性顆粒官能基改質為醛基固定探針 ……………………… 53
4.7.1磁性顆粒表面醛基之定量 …………………………… 53
4.7.2固定在顆粒上卵白素之定量 ………………………… 53
4.7.3磁性顆粒改質為醛基固定探針之雜合效率………… 54
4.8非磁性顆粒鍵結5’P primer2 雜合之專一性探討……………56
4.8.1基因序列完全不同 ……………………………………56
4.8.2基因序列其中連續7個base 與5’P primer2互補…59
4.9四種不同改質顆粒雜合效率之比較………………………… 63
4.10顆粒表面改質為醛基固定5’amino primer2
其鍵結量與雜合效率 ………………………………… 69
4.11六種不同改質顆粒雜合效率之比較 ……………………… 78
第五章 結論與建議 ……………………………………………………… 80
參考文獻…………………………………………………………………… 83

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