ISOLATION and CHARACTERIZATION of PARTIAL FRAGMENT of MITOCHONDRIAL GENOME (12S Rrna - ND2') of RASBORA SARAWAKENSIS
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"-0 1 E ý W . I . ý . e ' ý " ISOLATION AND CHARACTERIZATION OF PARTIAL FRAGMENT OF MITOCHONDRIAL GENOME (12S rRNA - ND2') OF RASBORA SARAWAKENSIS ALICE LAU KUI YAN (46219) Bachelor of Science with Honours (Resource Biotechnology) 2017 Isolation and Characterization of a Partial Fragment of Mitochondrial Genome (12S rRNA - ND21 from Rasbora sarawakensis ALICE LAU KUI YAN (46219) A thesis submitted in partial fulfilment of the requirements for the degree of Bachelor of Science with Honours (Resource Biotechnology) Supervisor: DR CHUNG HUNG HUI l Bachelor of Science (Honours) Resource Biotechnology Department of Molecular Biology Faculty of Resource Science and 'Technology University Malaysia Sarawak 2017 ACKNOWLEDGEMENT First and foremost, I would like to thank God for granting me strength, ability and opportunity to undertake and complete this research study. Without His blessing, this achievement would not have been possible. Next, I would like to express my thanks and endless appreciation to my supervisor, Dr. Chung Hung Hui for his continued guidance and support throughout the project. I have been extremely lucky to have a supervisor who cared so much about my work. He has been there providing his valuable knowledge and advices at all times to ensure I always stay on the core of my research. I would also like to thank Dr. Lee Kul Soon for their kindness to allow me to use the machine in their laboratories. I have great pleasure in acknowledging my gratitude to post-graduate students in Animal Biotechnology Lab, Aimi and Leonard. Their countless guidance and encouragement had greatly contributed to the completion of the thesis. Besides that, I would like to thank my lab mates, Woan Ting, Chew Pei Ling, Syahril, Chung Pey Lin, Lee Ying, Vivien, Abigail and Fatin for giving me various forms of assistance and motivation during the hard times. I would also likVo express my gratitude to my friends, Zi Wei, Kai Jia, Caren, Chloe, Jinny, Jia Min, Jeffrey, Kang Young, William and Woei Hann for their moral support and motivation. My acknowledgement would be incomplete without giving a special thanks to my family members: my dad Lau Hieng Yioung, any mum Shu How Ming, my sisters Agnes and Grace and my brother Andrew for their undying love. Their prayers for me were what sustained me thus far. I UNIVERSITI MALAYSIA SARAWAK Grade: Please tick ( ) Final Year Project Report 0 Masters I PhD 0 DECLARATION OF ORIGINAL WORK This declaration is the day 2017 made on ................. of................. Student's Declaration: I Alice Lau Kui Van, 46219, Faculty of Resource Science and Technology hereby declare that the work entitled, Isolation and Characterization of Partial Fragment of Mitochondrial Genome (12S rRNA -ND21 of Rasbora sarawakensis is my original work. I have not copied from from any other students' work or any other sources with the exception where due reference or acknowledgement is made explicitly in the text, nor has any part of the work been written for me by another person. I I /W'(7 elL-t- Date submitted Alice Lau Kui Yan (46219) Supervisor's Declaration: 1, Dr. Chung Hung Hui hereby certify that the work entitled Isolation and Characterization Partial Fragment Mitochondrial Genome of of (12S rRiVA - ND21 of Rasbora by sarawakensis was prepared the aforementioned or above mentioned student, and was * fulfillment submitted to the "FACULTY" as a partial/full for the conferment of Bachelor of Science with Honours (Resource Biotechnology), and the aforementioned work, to the best of my kno%Nledge. is the said students work Received for examination by: Date: (Dr. Chung Hung Hui) 11 I declare that Project/Thesis is classified as (Please tick (v)): ýJ CONFIDENTIAL (Contains confidential information under the Official Secret Act 1972)* RESTRICTED (Contains restricted information as specified by the done)* organisation where research was . OPEN ACCESS Validation of Project/Thesis I hereby duly affirmed with free consent and willingness declared that this said Project/Thesis shall be placed officially in the Centre for Academic Information Services with the abide interest and rights as follows: " This Project/Thesis is the sole legal property of Universiti Malaysia Sarawak (UNIMAS). " The Centre for Academic Information Services has the lawful right to make copies of the Project/Thesis for academic and research purposes only and not for other purposes. " The Centre for Academic Information Services has the lawful right to digitize the content to be uploaded into Local Content Database. for " The Centre Academic Information Services has the lawful right to make copies if of the Project/Thesis required for use by other parties for academic purposes or by other Higher Learning Institutes. " No dispute or any claim shall arise from the student himself / herself neither a third party on this Project/Thesis once it becomes the sole property of UNIMAS. " This Project/Thesis or any material, data and information related to it shall not be distributed, published or disclosed to any party by the student himself/herself without first obtaining approval from UNIMAS. Student's signature Supervisor's signature: (Date) (Date) Current Address: Notes: * If the Project/Thesis is CONFIDENTIAL or RESTRICTED, please attach together as annexure a letter from the organisation with the date of restriction indicated, and the reasons for the confidentiality and restriction. [The instrument was prepared by The Centre for Academic Information Services] III Table of Contents Content Page Acknowledgement .................................................................. I Declaration II ......................................................................... Table Contents IV of ................................................................ List Abbreviations of ............................................................ VI List Figure of ..................................................................... VII List Tables VIII of ..................................................................... Abstract IX .......................................................................... 1.0 Introduction 1 .................................................................. 1.1 Background 1 .......................................................... 1.2 Objectives ............................................................ 3 2.0 Literature Review 4 2.1 Mitochondrial genome ............................................. 4 2.2 Phylogenetic analysis ................................................ 7 2.3 Taxonomy classification .............................................. 10 2.4 Ciprinidae family ................................................... 11 2.5 Rasbora genus 13 ............ .............................................. 2.6 Rasbora Sarawakensis ............................................ 14 3.0 Materials Methods and ..................................................... 16 3.1 Sample collection and preparation 16 ............................... 3.2 Total DNA from Rasbora 16 extraction adult sarawakensis ........ 3.3 Agarose gel electrophoresis ......................................... 17 3.4 Primer design 18 .......................................................... 3.5 Gradient PCR ......................................................... 19 3.6 Purification PCR amplified 20 of products ........................... 3.7 DNA Sequencing and sequence analysis 21 .......................... 3.8 Phylogenetic analysis ................................................. 21 4.0 Result 23 ........................................................................... 4.1 Total DNA extraction ................................................ 23 4.2 Primer design and synthesis ........................................ 24 4.3 Temperature by PCR 25 optimization of primers gradient ......... IV 4.4 DNA 28 purification ...................................................... 4.5 Sequence 29 analysis .................................................... 4.6 Phylogenetic tree 32 ..................................................... 5.0 Discussion 33 ................................................... 5.1 Sequence 34 analysis ....................................................... 5.2 Phylogenetic analysis ................................................... 37 6.0 Conclusion ....................................................................... 39 7.0 Reference 40 ...................................................................... Appendix A ......................................................................... 45 Appendix B ......................................................................... 49 Appendix C ......................................................................... 50 Appendix D ......................................................................... 51 Appendix E ......................................................................... 52 Appendix F ......................................................................... 53 Appendix G ......................................................................... 54 Appendix H .......................................................................... 57 Appendix I ........................................................................... 58 V List of Abbreviations ATP Adenosine triphosphate BLAST Basic Local Alignment Search Tool bp Base pair CO! Cytochrome oxidase subunit I CSB Conserved Sequence Blocks D-loop Displacement loop DNA Deoxyribonucleic Acid mL Mililitres NADH Nicotinamide adenine dinucleotide