Design And Preliminary Testing Of A Novel Cartridge For The Capture And Containment Of Biomolecules

Abstract

Purification of biomolecules including DNA, RNA and protein from any biological material is the initial step prior to any downstream manipulation processes, such as DNA sequencing, RNA microarray and immunoblotting in molecular biology studies. Generally, there are two main nucleic acid purification methodologies - conventional and the solid phase extraction methods. The solid phase nucleic acid extraction method uses spin column and repeated centrifugation processes for extraction and purification. Most of the conventional extraction methods employ nonenvironmental friendly solvents such as ethanol or isopropanol, and chaotropic salts such as guanidinium thiocyanate. A new, rapid and easy solid phase extraction method without the use of organic solvent was designed to overcome the above problems. The solid phase used in this study was made of cellulose powder, a biodegradable material. Similar extraction protocol was used in the newly designed portable extraction system. Optimisation of the extraction process was performed using solution DRoP1 + DRoP3 and various types of microfiber materials as solid phase of the spin column. Gramnegative bacterium - Escherichia coli was used as the model organism for biomolecules extraction. Physical characterisation of the filter discs was performed to determine the consistency of the different batches of filter discs produced. The intact and defective filter discs were compared under scanning electron microscope. Up to 150 ng/μL of plasmid DNA was obtained from 1 mL of bacteria culture using the optimized centrifugation based extraction method, with high nucleic acid purity at OD 260/280 and OD 260/230 ratios, in the range of 1.7 to 2.12. The optimised extraction method was employed for the genomic DNA, RNA and proteins extraction from bacteria, plant, fungi and mammalian cell line. Genomic DNA (100 - 250 ng/μL) and RNA (10 – 90 ng/μL) were obtained from these organisms. The extracted plasmid DNA products were usable for polymerase chain reaction and restriction endonuclease digestion. Besides, RNA extracted from mammalian cell line was usable for reverse transcription polymerase chain reaction. On the other hand, protein extraction using this column reduced the viscosity of protein samples after adding the lysis buffer. In conclusion, a new biomolecules extraction method has been developed using microfiber material as centrifugation based and portable based cartridge for the capture and containment of biomolecules.