利用單股核苷酸修飾之金奈米粒子偵測單核苷酸多型性之研究

Detection of single nucleotide polymorphisms using ssDNA-Au nanoparticles

徐巧齡(Chiao-Ling Hsu)

指導教授 ： 侯劭毅

國立臺北科技大學/工程學院/生物科技研究所/碩士(2007年)

摘要

單核苷酸多型性(SNP)是最普遍發生的一種遺傳變異，在基因組DNA中每隔幾百到幾千個鹼基就會發生。個人化醫療的概念是根源於這些獨特多型性的鑑定，在疾病診斷和治療上提供了一個機會。到目前為止，許多技術已經被發展出來作為SNP的偵測方法。但是，這些傳統的方法相對耗時且成本較高。在這個研究中，我們希望能以單股特異性核酸酶和金奈米粒子為基礎的呈色分析系統，發展出一套簡單、低成本、且不需要精確溫控就能偵測完全互補的目標DNA分子與單一個鹼基配對錯誤的目標DNA分子間的差異。
這個SNP的偵測方法是經由三個步驟完成的，首先將目標DNA分子與assay probe雜交後的雙股DNA，利用mung bean nuclease在雜交雙股DNA中錯誤配對的位置處進行斷裂反應，而對於完全配對的雜交雙股DNA則不會有斷裂反應發生。反應後隨即加入capture Au-probe及anti-assay probe與上述酵素作用之產物反應，用離心的方式將互補於capture Au-probe的目標DNA回收下來。最後加入detector Au-probe和互補在capture Au-probe上的目標DNA分子進行反應，藉由全波長的掃瞄來分析目標DNA中是否有單一個配對錯誤的鹼基。
本研究所用的呈色分析系統可以偵測出樣本中在某個鹼基範圍內有SNP存在，但是至目前為止，還無法偵測到SNP確切的位置。

關鍵字

單核苷酸多型性(SNP) ； 金奈米粒子 ； 單股特異性核酸酶

並列摘要

Single nucleotide polymorphisms (SNP) are the most abundant form of genetic variation and occur as often as every few hundred to few thousand base pairs in genomic DNA. The concept of personalized medicine is rooted in the identification of these unique polymorphisms, providing opportunities in both the diagnosis and treatment of diseases. Up to now, many techniques have been developed for SNP detection. However, conventional methods are of relatively time-consuming and high cost. In this study, we develop a novel colorimetric detection system based on single-strand specific nucleases and gold nanoparticles, the assay provided a convenient yet powerful colorimetric detection that enabled a straightforward single-base discrimination without the need of precise temperature control.
The assay coluld be implemented via three steps: a hybridization reaction that allowed assay probe to hybrid with the target DNA strand, a cleavage reaction that generates between mismatched target and assay probe, but no cleavage reaction that generates between perfectly matched target and assay probe.When the reaction mixture was added capture gold-probe and anti-assay probe. After reaction, the solution by centrifugation to retrieve target DNA. Finally, the redispersed solution by adding a detector gold-probe.After reaction, the solution using a UV-vis spectrophotometer analysis was performed.
To our knowledge, this is the first report concerning SNP detection based on the single-strand specific nucleases and the gold nanoparticles assembly.

並列關鍵字

Single nucleotide polymorphisms ； gold nanoparticles ； single-strand specific

參考文獻

1. Sachidanandam, R., Weissman, D., Schmidt, S. C., Kakol, J. M., Stein, L. D., Marth, G., Sherry, S., Mullikin, J. C., Mortimore, B. J., Willey, D. L., “A map of human genome sequence variation containing1.42 million single nucleotide polymorphisms”, Nature, 2001, 409, 928–933.

2. Wabuyele, M. B., Farquar, H., Stryjewski, W., Hammer, R. P., Soper, S. A.,Cheng, Y. W., Barany, F., “Approaching real-time molecular diagnostics: single-pair fluorescence resonance energy transfer (spFRET) detection for the analysis of low abundant point mutations in K-ras oncogenes”, J. Am. Chem. Soc., 2003, 125, 6937–6945.

3. Sato, K, Onoguchi, M, Sato, Y, Hosokawa, K, Maeda, M., “Non-cross-linking gold nanoparticle aggregation for sensitive detection of single-nucleotide polymorphisms: Optimization of the particle diameter”, Analytical Biochemistry, 2006, 350, 162-164.

4. Woff, S. F., Haines, L., Fisch, J., Kremsky, J. N., Dougherty, J. P., Jacobs, K., “Rapid hybridization kinetics of DNA attached to submkron latex particles”, Nucleic Acids Res., 1987, 15, 2911-2926.

5. Weiss S., “Fluorescence spectroscopy of single biomolecules”, Science, 1999, 283, 1676-1683.