Design and optimization of strip test in examining kras mutation in colorectal cancer
Elaheh Ferdosi shahandashti,
1,* Elahe motevaseli,
2 Mohammad hossein modarressi,
3
1. Department of Medical Biotechnology, Faculty of Medicine, Babol University of Medical Science, Amol, Iran.
2. Tehran University of Medical Sciences
Abstract
Introduction
Kras mutations are among the key events in the carcinogenesis process of colorectal cancer. some mutations in the kras gene in metastatic colorectal cancer patients cause the patients to not respond to the monoclonal antibodies of panitumumab and cetuximab. therefore, colorectal cancer patients should be monitored for mutations in the kras gene before using this monoclonal antibody and only those with colorectal cancer that do not have mutations in the kras gene respond to these drugs and prevent tumor progression. in this study, the design of strip assay based on the reverse dot blot was used to investigate mutations of the kras gene in colorectal cancer patients.
Methods
In this study, 10 biopsy samples from healthy subjects and 10 biopsy samples from patients with colorectal cancer referred to imam khomeini hospital in tehran and ayatollah rouhani hospital in babol were collected. to design the strip test, nylon and nitrocellulose membranes were first selected and then 12asp, 12ala, wild-type kras and hla-control probes and kras and hla gene primers were designed. after membrane and probes treatment, probes hybridization was performed by biotinylated pcr products, derived from a patient and healthy specimen. reverse engineering was also used to design primers and probes, for which ta cloning technique was used. in the final step, to evaluate the results, the concentration and time of using the alkaline phosphatase enzyme attached to streptavidin and the bcip-nbt substrate were optimized to create the lowest color of the field for better resolution of the bands.
Results
in this study, 12asp, 12ala, wild-type kras and hla-control probes were coded with the appropriate and treated membrane stripes to survey mutations in the kras gene. then, the results of hybridization with pcr-labeled product of patient and healthy samples were analyzed separately. as an enzyme and substrate control, instead of a probe, the biotinylated product of pcr and to control the correct hybridization, instead of the probe, the non- biotinylated product of pcr was encoded on the strips. as a result, after each test, these two controls were observed as purple bands showing the correctness of the test. to control of pcr, an hla-control probe was used which was observed in a purple band in all tests, both in healthy samples and in patient samples, which shows the correctness of the pcr. in the case of healthy and patient specimens with 12asp, 12ala mutations, the wild-type kras, 12asp, and 12ala probe bands, in addition to the controls mentioned, were observed to be blue-purple violet. at all stages after optimization, favorable results were obtained.
Conclusion
The final result of this study was the design of a diagnostic kit for the study of mutation in colorectal cancer patients. the kit buffers, the appropriate membrane and 4 oligonucleotide probes were designed and optimized for temperature, time, concentration and ph, and favorable results were obtained. regarding different methods for kras gene analysis, the reverse dot blot method, which was designed in this study, has some advantages. this method has high sensitivity in detecting tumor cells and data analysis is easy to do, unlike other methods. hoping to acquire this knowledge, we can look at other cancer factors as well as various genetic diseases in the near future.
Keywords
Colorectal cancer, strip test, probe modification, kras
