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: Applications of genetics. Second Edition. John Murray (Publishers) Ltd. 4. Nicholl, D. S. T. (1994) An Introduction to Genetic Engineering. Cambridge University Press. 5. Weaver R. F. (2005) Molecular Biology, Chapter 24: Genomics and Proteomics. Third Edition. McGraw-Hill International Edition.

NANYANG JUNIOR COLLEGE H2 Biology Isolating, Cloning and Sequencing DNA J2/2016 3 1. Introduction Biotechnology (technology based on biology) is the manipulation of living systems and organisms to make and develop useful products. It includes early practic es such as selective breeding of farm animals and using microorganisms to make wine and cheese. Encompassed within biotechnology is genetic engineering, which is the direct manipulation of genes for practical purposes. Applications of genetic engineering include the manufacture of protein products, such as hormones. The sequencing of the human genome was largely completed by 2003.The potential benefits of the Human Genome Project are numerous yet there are ethical and moral implications behind many of the benefits. This topic covers the basic molecular techniques required for genetic engineering and how cloning of useful genes can lead to production of human proteins using bacteria cells. The goals, benefits and ethical issues surrounding the Human Gen ome Project will also be discussed. 2. Basic Molecular Techniques in Genetic Engineering (a) Restriction and Ligation (i) Restriction Enzymes (also called restriction endonucleases)  Naturally found in bacteria.  Protect the bacterial cells from other organisms or p hages by cutting up foreign DNA, a process known as restriction.  DNA of a bacterial cell is protected from the cell′s own restriction enzymes  Addition of methyl groups ( -CH3) to adenines or cytosines within the sequences recognised by the enzymes.  Very specific in action  Recognises a short specific DNA sequence known as the restriction site (usually four to eight nucleotides long).  Active site of the restriction enzyme is complementary to the three - dimensional structure of the specific nucleotide sequence.  Most restriction sites are palindromic, i.e. the sequence of nucleotides is the same on both strands read in the 5’  3’ direction.  Hydrolyse phosphodiester bonds in the sugar -phosphate backbone of both DNA strands at specific points within restriction sites.  Some restriction enzymes produce sticky ends while others produce blunt ends.

NANYANG JUNIOR COLLEGE H2 Biology Isolating, Cloning and Sequencing DNA J2/2016 4  Fragments with sticky ends have at least one single –stranded end because the two DNA strands are cut in a staggered manner. The single -stranded DNA can form hydrogen bonds with complementary sticky ends on any other DNA molecules cut with the same enzyme . E.g. EcoRI, BamHI and HindIII restriction enzymes cut DNA into fragments with sticky ends.  Fragments with blunt ends are produced when the two DNA strands are cut at the same specific position within the restriction site. (ii) DNA Ligase  Catalyses formation of phosphodiester bonds between two nucleotides.  Used after fragments cleaved using the same restriction enzyme are incubated together to produce a stable recombinant DNA molecule  Recombinant DNA refers to manipulated DNA molecules containing DNA from two or more sources  Complementary sticky ends form temporary hydrogen bonds after which DNA ligase is added to form a permanent phosphodiester bond.

NANYANG JUNIOR COLLEGE H2 Biology Isolating, Cloning and Sequencing DNA J2/2016 5 Formation of recombinant DNA molecule using restriction enzyme and DNA ligase

NANYANG JUNIOR COLLEGE H2 Biology Isolating, Cloning and Sequencing DNA J2/2016 6 (b) Addition of sticky ends to DNA molecules with blunt ends In genetic engineering, when restriction fragments have blunt ends, additional steps are required to form complementary stick y ends . E.g. HaeIII and AluI restriction enzymes cut DNA into fragments with blunt ends. (i) Addition of specific linker DNA  Specific linker DNA is a short, synthetic double-stranded oligonucleotide which contains a specific restriction site of a particular restriction enzyme.  Added to both the blunt ends of the DNA fragment using DNA ligase  Linker DNA is then further cut by appropriate restriction enzyme to produce sticky ends. Addition of specific linker DNA to form sticky ends (ii) Use of terminal transferase  Terminal transferases are enzymes which catalyse addition of deoxyribonucleotides (more specifically deoxyribonucleoside triphosphates) at the 3’ ends of a DNA molecule without using a template.  Deoxycytidine triphosphate (dCTP) can be added to one blunt -ended DNA fragment and deoxyguanosine triphosphate (dGTP) can then be added to the other DNA fragment to produce complementary sticky ends.