2016_H2_Topic_O_Isolating__Cloning_and_Sequencing_Lecture

NANYANG JUNIOR COLLEGE H2 Biology Isolating, Cloning and Sequencing DNA J2/2016 1 TOPIC O: ISOLATING, CLONING AND SEQUENCING DNA Learning Outcome Applications Topic 6 Candidates should be able to: (a) Describe the natural function of restriction enzymes. (b) Explain the formation of recombinant DNA molecule. (c) Outline the procedures for cloning a eukaryotic gene in a bacterial plasmid and describe the properties of plasmids that allow them to be used as DNA cloning vectors. (d) Distinguish between a genomic DNA and cDNA library. (Outline of the process of the formation of the libraries and applications of each of the types of library is required.) (e) Explain how eukaryotic genes are cloned using E. coli cells to produce eukaryotic proteins to avoid the problems associated with introns. (f) Outline two important proteins that can be produced by genetic engin eering technique (e.g. human growth hormone and insulin). (g) Describe the polymerase chain reaction (PCR) and explain the advantages and limitations of this procedure. (h) Explain how gel electrophoresis is used to analyse DNA. (i) Outline the process of nucleic acid hybridization and explain how it can be used to detect and analyse restriction fragment length polymorphism (RFLP). (j) Explain how RFLP analysis facilitated the process of : i. genomic mapping in terms of linkage mapping; ii. diseases detection, e.g. sickle cell anaemia; iii. DNA fingerprinting. (Details of application of gel electrophoresis, PCR and nucleic acid hybridisation in RFLP may be required.) (k) Discuss the goals and implications of the Human Genome Project, including the benefits and difficult ethical concerns for humans. (Knowledge of the te chnical procedure of the Human Genome Project and DNA sequencing is not required.)

NANYANG JUNIOR COLLEGE H2 Biology Isolating, Cloning and Sequencing DNA J2/2016 2 Content Outline 1. Introduction 2. Basic Molecular Techniques in Genetic Engineering (a) Restriction and Ligation (b) Addition of sticky ends to DNA molecules with blunt ends (c) Polymerase Chain Reaction (d) Gel Electrophoresis (e) Nucleic Acid Hybridisation 3. DNA Libraries (a) Genomic Library (b) Complementary DNA Library 4. Gene Cloning (a) Plasmids (b) Process of Gene Cloning (c) Production of Proteins using Genetic Engineering (d) Other Products of Genetic Engineering 5. Restriction Fragment Length Polymorphism Analysis (a) Disease Detection (b) DNA Fingerprinting (c) Genome Mapping 6. Human Genome Project (a) Goals (b) Benefits (c) Ethical Issues References 1. Brooker, R. J. (2005) Genetics: Analysis an d Principles. Chapter 18: Recombinant DNA Technology. Second Edition. McGraw-Hill. 2. Campbell, N. A. and Reece J. B. (2011 ) Biology. Chapter 20: Biotechnology. Ninth Edition. Pearson Education. Inc. 3. Clegg, C. J. and MacKean, D. G. (2000) Advanced Biology: P rinciples and Applications. Chapter 29: Application