Abstract
Development of an Optimized Protocol for Human DNA Extraction and PCR Amplification of the CtsK Gene for Downstream Development of Biopharmaceuticals, Enzyme Inhibitors and Inducers For Cathepsin K
Madubashetha, H., Wickramasinghe, S., De Silva, N., Warnakula, L., & Cooray, R.
17th – 18th October 2019
Steering Horizons; Aspiring Excellence!, International Conference on Health Sciences 2019, University of Sri Jayewardenapura, Colombo, Sri Lanka.
Proceedings of the International Conference on Health Sciences 2019, (pp. 153).
doi:
Background: Cathepsin K, encoded by CtsK gene involves in bone remodelling through ossification. Despite this orthopaedic importance, it demonstrates physiological importance including metastasis of prostate, ovarian and breast cancers. It is a timely concern that inhibitors or inducers that stimulate or inhibit activity of Cathepsin K are brought to arena of pharmaceuticals with the recombinant production of Cathepsin K.
Objectives: To develop an optimized protocol for isolation of DNA from human blood and PCR amplification of CtsK gene facilitating downstream production of Cathepsin K biopharmaceuticals by molecular characterization of CtsK gene.
Methods: Genomic DNA was extracted from 04 human blood samples using FlexiGene®-QIAGen®, subjecting blood to action of cell lysis, denaturation and resuspension buffers. Incubation times and number of 70% ethanol washing times were increased and spectrophotometric absorbency was measured. PCR amplification of CtsK gene using primers 5’ACGCGTCGACTTAATTCCATGGTTAGTTCCCC’3 and 5’ACGCAAGCTTGGTCATGCCAGATTACATATGC’3 was done. PCR conditions were optimized; 94°C initial denaturation, 94°C denaturation, 55°C annealing, 72°C elongation, 72°C final elongation and 4°C final hold for 3 minutes, 30 seconds, 30 seconds, 40 seconds, 5 minutes and infinite respectively.
Results: Accordingly, all DNA samples showed a concentration of nearly 500ng/µl while for purity, A260/A280 ratio for DNA was revealed to be between 1.7 and 1.8 while A260/A230 was revealed to be between 1.7 and 2.2, reflecting good purity. As a result of PCR, a band of size 550bp was generated in 1.5% TAE-agarose gel, which was verified to be a catalytic domain of Cathepsin K by previous literature that could now be directed towards cloning followed by expression.