期刊
JOURNAL OF CHROMATOGRAPHY A
卷 1218, 期 7, 页码 997-1003出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.chroma.2010.12.089
关键词
Multi-target; Fraction collection; Fractionation; Electrophoresis; Contamination; Isolated extraction channels
资金
- Japan Science and Technology Agency
- Ministry of Education, Culture, Sports, Science, and Technology of Japan [19049001, 470]
- Hokkaido Innovation through Nanotechnology Support (HINTS)
- Grants-in-Aid for Scientific Research [19049001] Funding Source: KAKEN
High efficiency and high-purity fraction collection is highly sought in analysis of fragments-of-interest from selective polymerase chain reaction (PCR) products generated by High Coverage Gene Expression Profiling (HiCEP) methods. Here we demonstrate a new electrophoretic chip device enabling automatic high-efficient fractionation of multiple ssDNA target fragments during a run of separation. We used thoroughly isolated extraction channels for each selected target to reduce the risk of cross-contamination between targets due to cross-talk of extraction channels. Fragments of 35, 108 and 138 b, were successfully isolated, then the recovery was PCR-amplified and assessed by capillary electrophoresis (CE) analysis. Total impurity level of the targets due to unwanted fragments of 0.7%, 2% and 6% respectively, was estimated. Difficulties in collecting multiple target factions are due to band diffusion and DNA adsorption to the walls for the fragments in the separation channel, which is generated by transferring the DNA target fraction from the extraction section to the target reservoir. Therefore, we have carefully measured band broadening and analyzed its influence on the separation resolution due to the delay. (C) 2010 Elsevier B.V. All rights reserved.
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