cDNA微陣列上探針點之表面形態研究

Surface Morphology Study of cDNA Microarray Probe Spots

鄭光泓(Kuang-Hong Cheng)

指導教授 ： 許志楧 ； 吳見明

國立清華大學/原子科學院/生醫工程與環境科學系/碩士(2008年)

https://doi.org/10.6843/NTHU.2008.00029

摘要

中文摘要
cDNA微陣列是使用一具三度空間的自動機械手臂系統，將聚合脢連鎖反應 （Polymerase Chain Reaction，簡稱 PCR）後之DNA產物以高密度點製在經表面處理過的基材（玻片）而成的微陣列。cDNA微陣列上的DNA片段，稱為探針（probe）。當探針和染有螢光的標的物進行雜合反應後，人們會對微陣列進行掃描並觀察探針點的螢光訊號，並從螢光訊號強弱判定基因表現量。然而探針點的DNA分佈會對基因表現量在量測上造成影響，所以研究微陣列上探針點的表面形態就有其重要性。本研究旨在以新的光譜影像擷取技術與分析方法，來研究cDNA微陣列上探針點的DNA分佈。
實驗上利用光譜影像掃描系統掃描微陣列上染有Cy3的探針點並得到其光譜。再利用光譜特性來判斷微陣列上的物質。例如：因為探針上的DNA染有Cy3，所以有Cy3的螢光訊號就表示有DNA，還有點印液SSC的結晶不會有螢光訊號，因此也不會對光譜訊號造成干擾。實驗上也將擷取微陣列在各標準實驗步驟下探針點的表面形態影像和螢光影像，並藉由觀察擷取的影像和光譜，進而研究探針點的形態分佈。由觀察結果顯示微陣列上的結晶分佈與DNA的分佈相關性達0.9以上。另外蒸濕作用（rehydration）和表面封阻（surface blocking）也都會對cDNA微陣列上探針點的表面形態分布有影響。其中在包含熱固定（snap-heating）、紫外光鍵結（UV-crosslinking）與烘乾（baking）的蒸濕作用後，SSC與DNA溶液會往外擴散並在外圍結晶，且結晶表面會析出DNA，部分DNA則會往內聚集，使得探針點變圓變大且DNA分佈更為均勻。再經過表面封阻與清洗後，未與cDNA微陣列玻片結合的DNA和SSC會被洗去，因此探針點上DNA分佈又會變得較不均勻。研究探針點上DNA分佈的均勻度會有助於cDNA微陣列在進行雜合反應後得到可靠的結果。

關鍵字

cDNA微陣列 ； 探針點 ； 表面形態研究

並列摘要

Abstract

cDNA microarray contains densely packed spots with known DNA sequences. The deposited DNA sample is referred to as the probe. After the fluorescently-labeled target DNA is hybridized to its complementary probe strand, the microarray was scanned to measure the intensity of the fluorescent signals on each spot. The intensity of each spot represents the expression level of a specific gene. However, the DNA distribution within each spot will influence the analysis of the fluorescent signals. This research aims at applying the spectral images technology and other analytical methods to study the distribution of DNA within the probe spot and analyze the cause of its irregular distribution.
The spectral-scanning system was applied in this study to scan the spectrum of probes labeled with Cy3 dye on the cDNA microarray. The characteristics of spectrum were used to distinguish the fluorescence, which may overlap with that of Cy3, emitted from the other materials on the cDNA microarray. SSC, which was added in the DNA spotting solution, do not produce fluorescent signal, therefore cannot be detected by the fluorescence and spectral imaging systems. The non- fluorescence morphologic image acquired by EMCCD camera represents the distribution of crystal, after the spotting solution was dry out. We compared the morphologic and fluorescent images of each spot. The pixel correlation between the distribution of DNA and crystal is higher than 0.9. Furthermore, data reveals that rehydration and surface blocking also affected the distribution of crystal and Cy3-labeled DNA on the glass surface. When microarray slides were processed by rehydration followed by snap-heating, UV cross-linking and baking, the SSC and DNA crystal diffused outward and crystallized out DNA on its surface. DNA molecules tend to gather inward, thus the spots become more rounded, enlarged and the DNA is more evenly distributed. After the surface blocking and washing, DNA molecules and SSC crystal that were loosely attached to the glass surface were washed away. Therefore, the distribution of DNA became less homogenized. The degree of homogenization of DNA distribution on the glass surface affects the reliability of cDNA microarray results.

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