Jiang Xinglu, Zhao Chenggui, Fan Xiaobo, Xu Wei, Zhang Rui, Xu Hongbo, Wu Guoqiu
Medical School of Southeast University Nanjing 210009 People's Republic of China
Center of Clinical Laboratory Medicine, Zhongda Hospital, Southeast University Nanjing 210009 People's Republic of China.
RSC Adv. 2019 Jan 25;9(6):3407-3415. doi: 10.1039/c8ra10138h. eCollection 2019 Jan 22.
Cell-free DNA (cfDNA) in blood, which stems from the fetus of pregnant women and tumor in cancer patients, has gained attention in molecular diagnosis. However, cfDNA is less stable, and its amount in the serum is extremely low; these are critical barriers for the utilization of this resource. In this study, a DNA-modified polyacrylamide hydrogel (DNA-Gel) was prepared, and a specialized device was designed to simultaneously catch, purify, concentrate, and detect targeted cfDNA by electrophoresis. We demonstrated that 20-1000 bp ssDNA and dsDNA could be caught and released by the DNA-Gel-based device with high specificity and sensitivity. Upon increasing the number of cycles and electrophoresis time, higher DNA purity and density were achieved, and the separation of serum proteins, untargeted cfDNA, and other charged molecules was promoted. As low as 10 pg μL of DNA could be detected using the DNA-Gel after four cycles of concentration. We also detected 1 fg μL of DNA in the serum with 16 cycles of concentration, followed by 25 PCR cycles. We also designed a device to obtain DNA from the DNA-Gel. We found that the DNA loss rate was around 50%, and A260/A280 was close to 1.7. Thus, we have designed a cost-effective and highly economical device to purify DNA at low concentrations with high specificity and selectivity.
血液中的游离DNA（cfDNA）来源于孕妇的胎儿和癌症患者的肿瘤，在分子诊断中受到关注。然而，cfDNA稳定性较差，血清中含量极低，这些是利用该资源的关键障碍。本研究制备了一种DNA修饰的聚丙烯酰胺水凝胶（DNA-Gel），并设计了一种专门装置，通过电泳同时捕获、纯化、浓缩和检测靶向cfDNA。我们证明基于DNA-Gel的装置能够以高特异性和灵敏度捕获并释放20 - 1000 bp的单链DNA和双链DNA。随着循环次数和电泳时间增加，可实现更高的DNA纯度和浓度，促进血清蛋白、非靶向cfDNA及其他带电分子的分离。经过四个浓缩循环后，使用DNA-Gel可检测低至10 pg/μL的DNA。经过16个浓缩循环及25个PCR循环后，我们还检测到血清中1 fg/μL的DNA。我们还设计了一种从DNA-Gel获取DNA的装置。我们发现DNA损失率约为50%，A260/A280接近1.7。因此，我们设计了一种经济高效的装置，能够以高特异性和选择性纯化低浓度DNA。
