Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology , Beijing 100029, China.
Department of Nephrology, Shenzhen Affiliated Hospital, Guangzhou University of Chinese Medicine , Shenzhen 518033, Guangdong, China.
Anal Chem. 2017 Aug 1;89(15):8084-8091. doi: 10.1021/acs.analchem.7b01599. Epub 2017 Jul 12.
Mutagenic modifications of nucleotides or DNA lesions that result from environmental stress have proven to be associated with a variety of diseases, particularly cancer. The method for accurately detecting the lesions is therefore of great importance for biomedical research and toxicity study. We develop a sensitive and low-cost bulge-enhanced coamplification at lower denaturation temperature polymerase chain reaction (COLD-PCR) method for detecting DNA lesions (uracil and 8-oxoguanine) by combining an in vitro base excision repair (BER) pathway and COLD-PCR. The modified bases are converted to bulge via the BER pathway involving converting modified bases to an apurinic/apyrimidinic (AP) site, cleavage at the AP site, and break ligation. The presence of the bulge induces a large change of the hybridization thermodynamics of double-stranded DNA, eventually enhancing the specificity of COLD-PCR. Besides, we used the free energy of hybridization as a reference to optimize the critical denaturation temperature (T) of COLD-PCR obtaining more specific amplification than empirical T. Taking advantage of the proposed bulge-enhanced COLD-PCR, we are able to identify the presence of DNA lesion-containing strands at low abundance down to 0.01%. This method also exhibits high sensitivity for glycosylase with a detection limit of 10 U/mL [3 S/N (signal-to-noise ratio)] that is superior than some recently reported methods. With the design of the repair guide probe, the level of oxidative damage in genomic DNA caused by chemicals and photodynamic therapy (PDT) can be evaluated, heralding more applications in clinical diagnosis and epigenetic study.
环境应激导致的核苷酸或 DNA 损伤的诱变修饰已被证明与多种疾病（尤其是癌症）有关。因此，准确检测损伤的方法对于生物医学研究和毒性研究非常重要。我们通过结合体外碱基切除修复（BER）途径和 COLD-PCR，开发了一种灵敏且低成本的膨出增强在较低变性温度下的聚合酶链反应（COLD-PCR）方法，用于检测 DNA 损伤（尿嘧啶和 8-氧鸟嘌呤）。通过 BER 途径将修饰的碱基转化为膨出，该途径涉及将修饰的碱基转化为无嘌呤/无嘧啶（AP）位点，AP 位点的切割，以及断裂连接。膨出的存在会引起双链 DNA 杂交热力学的巨大变化，最终增强 COLD-PCR 的特异性。此外，我们使用杂交自由能作为参考来优化 COLD-PCR 的临界变性温度（T），从而获得比经验 T 更具特异性的扩增。利用所提出的膨出增强型 COLD-PCR，我们能够以低丰度（低至 0.01%）识别含损伤链的存在。该方法还具有高灵敏度的糖苷酶，检测限为 10 U/mL [3 S/N（信噪比）]，优于一些最近报道的方法。通过修复指导探针的设计，可以评估化学物质和光动力疗法（PDT）引起的基因组 DNA 氧化损伤水平，预示着在临床诊断和表观遗传学研究中有更多的应用。
