Focal extraction of surface-bound DNA from a microchip using photo-thermal denaturation.
Focal extraction of surface-bound DNA from a microchip using photo-thermal denaturation.
复制标题
使用光热变性从微芯片中聚焦提取表面结合的 DNA。
DOI：
10.2144/00285rr06
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发表时间：
2000
期刊：
影响因子：
2.7
通讯作者：
Shin'ichi Ishiwata
中科院分区：
文献类型：
--
作者：
Kenji Yasuda;K. Okano;Shin'ichi Ishiwata
在DNA分析领域，高通量、选择性地在PCR扩增前从混合DNA中提取特定的DNA片段变得越来越重要。尽管最新的微芯片技术已经能够使用表面结合探针DNA和样本DNA之间的杂交进行实时DNA表达分析，但该技术在纯化少量DNA方面的潜力尚未得到证实。我们在这里报告了一种区域选择性释放和收集特定DNA的方法，其中红外激光束聚焦在目标斑点区域的表面结合样本DNA上以变性杂交DNA。首先，用荧光染料标记的样本DNA与固定在镀铬芯片上的探针DNA杂交。然后以10微米的空间分辨率将强度为10-100 mW的1053 nm红外激光束聚焦在目标区域，导致荧光团标记的样品DNA因光热变性而释放。收集后通过PCR扩增确认洗脱的DNA量，表明从芯片中洗脱的DNA超过10(-20)mol /micron 2，占芯片结合样品DNA的70%以上。结果表明，该方法可应用于微芯片技术中DNA的高灵敏度纯化。
High-throughput, selective extraction of a particular DNA fragment from a mixture of DNA before PCR amplification is becoming increasingly important in the DNA analysis field. Although the latest microchip technology has enabled real-time DNA expression analysis using hybridization between surface-bound probe DNA and sample DNA, the potential of this technology in purification of a small amount of DNA has not been demonstrated. We report here a method for area-selective release and collection of specific DNA, in which an IR laser beam is focused onto surface-bound sample DNA at the target-spotted area to denature hybridized DNA. First, sample DNA labeled with a fluorescent dye was hybridized to a probe DNA immobilized on a chromium-coated chip. A 1053-nm IR laser beam with an intensity of 10-100 mW was then focused on the target area with a spatial resolution of 10 microns, causing the release of the fluorophore-labeled sample DNA as a result of photo-thermal denaturation. Confirmation of the amount of eluted DNA by PCR amplification after collection indicated that more than 10(-20) mol DNA/micron 2 area was eluted from the microchip, representing more than 70% of the chip-bound sample DNA. These results indicate that this method can be applied to the highly sensitive purification of DNA in microchip technology.