A DNA-modified hydrogel for simultaneous purification, concentration and detection of targeted cfDNA in human serum
2019
Jiang, Xinglu | Zhao, Chenggui | Fan, Xiaobo | Xu, Wei | Zhang, Rui | Xu, Hongbo | Wu, Guoqiu
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⁻¹ of DNA could be detected using the DNA-Gel after four cycles of concentration. We also detected 1 fg μL⁻¹ 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.
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Эту запись предоставил National Agricultural Library