Coupling technique of random amplified polymorphic DNA and nanoelectrochemical sensor for mapping pancreatic cancer genetic fingerprint

Abstract

Qicai Liu1,2, Ailin Liu3,4, Feng Gao5, Shaohuang Weng3,4, Guangxian Zhong3,4, Jingfeng Liu6, Xinhua Lin3,4, Jian-hua Lin7, Xuhai Chen81Department of Laboratory Medicine, Fujian Medical University, Fuzhou, 2Department of Gene Diagnosis, Fujian Medical University, Fuzhou, 3Department of Nano-Biomedical Research Center, Fujian Medical University, Fuzhou, 4Department of Pharmaceutical Analysis, Fujian Medical University, Fuzhou, 5Department of Pathology, Fujian Medical University, Fuzhou, 6Department of Surgery, Fujian Medical University, Fuzhou, 7Department of Bone Oncology, First Affiliated Hospital, Fujian Medical University, Fuzhou, 8College of Electrical Engineering and Automation, Fuzhou University, Fuzhou, People's Republic of ChinaObjective: To review the feasibility of coupling the techniques of random amplified polymorphic DNA (RAPD) with carbon nanotube-based modified electrode for guanine/deoxy-guanine triphosphate (dGTP) electrochemical sensing for mapping of the pancreatic cancer genetic fingerprint and screening of genetic alterations.Methods: We developed a new method to study the electrochemical behavior of dGTP utilizing carbon multiwalled nanotube (MWNT)-modified glassy carbon electrodes (GCEs). RAPD was applied for amplification of DNA samples from healthy controls and patients with pancreatic cancer under the same conditions to determine the different surplus quantity of dGTP in the polymerase chain reaction (PCR), thereby determining the difference/quantity of PCR products or template strands. Using this method we generated a genetic fingerprint map of pancreatic cancer through the combination of electrochemical sensors and gel electrophoresis to screen for genetic alterations. Cloning and sequencing were then performed to verify these gene alterations.Results: dGTP showed favorable electrochemical behavior on the MWNTs/GCE. The results indicated that the electrical signal and dGTP had a satisfactory linear relationship with the dGTP concentration within the conventional PCR concentration range. The MWNTs/GCE could distinguish between different products of RAPD. This experiment successfully identified a new pancreatic cancer-associated mutant gene fragment, consisting of a cyclin-dependent kinase 4 gene 3' terminal mutation.Conclusion: The coupling of RAPD and nanoelectrochemical sensors was successfully applied to the screening of genetic alterations in pancreatic cancer and for mapping of DNA fingerprints.Keywords: nanoelectrochemical sensor, random amplified polymorphic DNA, genetic fingerprint, pancreatic cancer, genetic predisposition, carbon nanotube