BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies.
BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies.
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BTA 是一种新型试剂，用于将 DNA 附着在玻璃上并有效生成固相扩增 DNA 菌落。
DOI：
10.1093/nar/gnj023
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发表时间：
2006-02-09
影响因子：
14.9
通讯作者：
Turcatti, G
中科院分区：
文献类型：
--
作者：
Fedurco, M;Romieu, A;Williams, S;Lawrence, I;Turcatti, G
使用三羧酸试剂苯-1,3,5-三乙酸（BTA）将5 ' -胺化DNA引物和模板附着在氨基硅化玻璃表面上，用于原位固相扩增下一代DNA菌落。我们对衍生化表面进行了表征，并评估了与扩增过程相关的性质：表面密度、热循环的热稳定性、功能化再现性和储存稳定性。衍生化工艺最初是为玻璃载玻片开发的，后来被应用于包含集成流体连接的微制造玻璃通道。这种实现大大减少了反应时间、试剂消耗和过程自动化。开发了新的附着DNA表征分析方法，用于评估扩增后表面固定DNA的含量。结果表明，BTA的化学性质是相容的，适合于从固定的初始单模板DNA分子扩增形成高密度的DNA菌落阵列，其最佳表面覆盖率约为1000万菌落/cm2。我们还证明了产生的dsDNA菌落可以通过限制性内切酶在原位定量处理。使用BTA试剂生成的DNA菌落可用于进一步的序列分析，以前所未有的平行方式进行低成本基因组研究。
The tricarboxylate reagent benzene-1,3,5-triacetic acid (BTA) was used to attach 5′-aminated DNA primers and templates on an aminosilanized glass surface for subsequent generation of DNA colonies by in situ solid-phase amplification. We have characterized the derivatized surfaces for the chemical attachment of oligonucleotides and evaluate the properties relevant for the amplification process: surface density, thermal stability towards thermocycling, functionalization reproducibility and storage stability. The derivatization process, first developed for glass slides, was then adapted to microfabricated glass channels containing integrated fluidic connections. This implementation resulted in an important reduction of reaction times, consumption of reagents and process automation. Innovative analytical methods for the characterization of attached DNA were developed for assessing the surface immobilized DNA content after amplification. The results obtained showed that the BTA chemistry is compatible and suitable for forming highly dense arrays of DNA colonies with optimal surface coverage of about 10 million colonies/cm2 from the amplification of initial single-template DNA molecules immobilized. We also demonstrate that the dsDNA colonies generated can be quantitatively processed in situ by restriction enzymes digestion. DNA colonies generated using the BTA reagent can be used for further sequence analysis in an unprecedented parallel fashion for low-cost genomic studies.
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