TECHNIQUES IN ANSWERING BIOCHEMICAL QUESTIONS, WITH SPECIAL REFERENCE TO NUCLEIC ACIDS Larissa Assam (SUNY Oswego)

1. TECHNIQUES IN ANSWERING BIOCHEMICAL QUESTIONS, WITH SPECIAL REFERENCE TO NUCLEIC ACIDS Larissa Assam (SUNY Oswego) Dr. DhrubajyotiChattopadhyay (University of Calcutta) INTRODUCTION ABSTRACT Analysis of deoxyribose nucleic acid (DNA) is essential in determining the genealogy of an organism. In order to study the nucleic acids, a mastery of the different techniques for handling DNA is essential because that will allow one to choose a technique for the required data. These experiments are aimed at obtaining a mastery of the techniques required for handling nucleic acids. This project was aimed at gaining a mastery in techniques necessary for handling nucleic acids. These techniques were used to determine the genealogy of bacteria from soil samples obtained from the banks of the River Ganges in the Sunderbans forest area. The desired DNA sequence was inserted into plasmid DNA and replicated in E.coli cells. An Agarose gel was run to see if the recombinant DNA was effectively removed from the host cells, the DNA was amplified by PCR and another gel was done to ensure that the PCR products were of the same size. The BLAST search based on 16S rRNA gene sequences indicated that the bacteria present in the soil sample included: Gramellaportivictoriae, Gramellaechinicola, Bacillus decolorationis and Microbulbiferdonghaiensis. WORK FLOW: METHODS and TECHNIQUES 3. Plasmid Isolation Cloning (previously executed) Vector: Target gene: 16S rRNA Endonucleases 2. Transformation Host: E.coli cells Conditions: Heat shock: 30 minutes at 4°C → 90 seconds at 37°C→5 minutes at 4 °C, 37°C overnight incubation Measurement of isolated DNA concentrations using Nanodrop Spectrophotometry Transformation of recombinant DNA Isolationof recombinant DNA from E.coli cells Measurement of isolated DNA concentrations using UV Spectrophotometry 5.Measuring DNA concentrations b. Nanodrop Spectrophotometry 6. Visualization of Isolated recDNA in agarose gel electrophoresis DNA sequencing of recombinant DNA gene (PCR) Amplification of recombinant DNA gene (PCR) Visualization of recDNA isolation in agarose gel electrophoresis Visualization of PCR products in agarose gel electrophoresis 260/280 ratio: purity of the sample 260/230 ratio: presence of organic solvents 4. Plasmid Purification 5.Measuring DNA concentrations a. UV Spectrophotometry measuring concentrations of recDNA extracted Concentration (µg/ml)= A260 x Dilution factor x (50 µg/ml/1 A260) Denaturation Denaturation Annealing (30 Cycles) Annealing (30 Cycles) Elongation Elongation 94 °C 96 °C 94 °C 96 °C C1= 70 µg/ml C4= 55 µg/ml C5= 60 µg/ml C6= 175 µg/ml 7. Polymerase Chain Reactions CONCLUSION 72 °C 60 °C 5’ 5’ Standard (Normal PCR) 4 °C 72 °C 8. Visualization of standard PCR products in agarose gel electrophoresis 9. DNA Sequencing 30” 10” The 16S rRNA BLAST search revealed 4 species of bacteria which are all aerobic and marine. There are several species of bacteria present in the Sunderbans forest area. Further work could be done to study the genomic properties of this bacteria and also on indentifying unknown species. 7‘ 7 minutes 1’ 4’ 52 °C 50 °C b) Nanodrop Spectrophotometry BLAST search: 16S rRNA database Bacteria found: Microbuliferdonghaensis Bacillus decolorationis Gramellaechinicola Gramellaportivictoriae 10” 40” REFERENCES ACKNOWLEDGEMENTS Lau, S. C., Wong , P.-K., Tsoi, M., Li, X., Plakhotnikova, I., Dobretsov, S., et al. (2005). Gramellaportivictoriae sp. nov., a novel member of the family Flavobacteriaceae isolated from marine sediment. International Journal of Systematic and Evolutionary Microbiology, 2497-2500. Journal of Systematic and Evolutionary Microbiology, 2497-2500. Old, R., & Primrose, S. (1994). Principles of Gene Manipulation. Blackwell Science. I will like to thank SUNY Oswego and Global Lab for this opportunity and for funding. I will also like to thank all the members of Dr. Chattopadhyay’s lab for their guidance and assistance throughout this project.