TMJC Molecular Technique in DNA Analysis Notes

Page 1 of 27 Tampines Meridian Junior College JC1 H2/9744 Biology 2023 Core Idea 2C 7. Genetics & Inheritance (IV) – Molecular Techniques in DNA Analysis SYLLABUS OVERVIEW No. Overarching Idea Topics 1 Core Idea 1 The Cell and Biomolecules of Life Cell – The Basic Unit of Life 2 Biomolecules of Life and Cellular Transport 3 Core Idea 3 Energy and Equilibrium Transformation of Energy – Photosynthesis and Cellular Respiration 4 Core Idea 2 Genetics and Inheritance Genetics and Inheritance (I) – The Cell Cycle 5 Genetics and Inheritance (II) – DNA Replication and Gene Expression 6 Genetics and Inheritance (III) – DNA Mutations and their Consequences 7 Genetics and Inheritance (IV) – Molecular Techniques in DNA Analysis 8 Genetics and Inheritance (V) – Organization of Genome & Control of Gene Expression in Eukaryotes [Includes Core Idea 1D: Stem Cells] 9 Genetics and Inheritance (VI) – Organization and Inheritance of Viral Genomes 10 Genetics and Inheritance (VII) – Organization of Genome & Control of Gene Expression in Prokaryotes 11 Genetics and Inheritance (VIII) - Inheritance 12 Core Idea 3 Energy and Equilibrium Communication and Equilibrium in Multicellular Organisms 13 Core Idea 4 Biological Evolution Biological Evolution 14 Extension Topic A Infectious Diseases Immunity and Infectious Diseases 15 Extension Topic B Impact of Climate Change on Animals & Plants Climate Change – Causes and Impacts on Animals and Plants

Page 2 of 27 NARRATIVES An understanding of Genetics and Inheritance that would help make sense of the transition from molecular to organismal level. Genetics and Inheritance provides the molecular basis to the understanding of how variations in populations arise and this is important in the study of biological evolution. At the cellular level, expression of genes involves cellular structures such as the nucleus, endoplasmic r eticulum and ribosome. Many essential products of gene expression are enzymes involved in biochemical pathways which control physiological functions. As such, mutation of genes may give rise to dysfunctional proteins which in turn could result in diseases. Sickle cell anemia and cancer are raised as examples of a monogenic and a mul ti-genic disease respectively. The following questions should help students frame their learning: • How does the genetic make-up of an organism influence its appearance, behavior and survival? • How can we ensure continuity of human as a species? Heritable information, in the form of DNA (and in some cases RNA), provides for continuity of life Genetic information is stored in an organism’s DNA; expression of genes results in the synthesis of functional products, such as rRNA, tRNA and proteins. These products play a role in intra- and extra-cellular biochemical pathways and influence the physiological processes in organisms. Genomes contain heritable information necessary for continuity of life at all levels: cell, organism and system. This information is stored and passed on to subsequent generations via DNA. Reproduction can occur at the cellular or organismal level; each progeny needs to receive heritable genetic information from its parents. Gene expression can be studied using fundamental techniques of molecular biology such as the polymerase chain reaction (PCR), gel electrophoresis, Southern blotting and nucleic acid hybridization. LEARNING OUTCOMES Core Idea 2C: Control of Gene Expression Basic molecular techniques allow scientists to study gene expression. Candidates should be able to: c) Describe the principles and procedures of these molecular techniques: i) polymerase chain reaction (including its advantages and limitations); ii) gel electrophoresis; and iii) Southern blotting and nucleic acid hybridization

Page 3 of 27 LECTURE OUTLINE 0. Overview of molecular techniques used in DNA analysis 1. Polymerase chain reaction 1.1 Introduction 1.2 Principles and procedures 1.3 Advantages and limitations of PCR 1.4 Applications (FYI) 2. Gel electrophoresis 2.1 Introduction 2.2 Principles and procedures 2.3 Applications (FYI) 3. Southern blotting and nucleic acid hybridization 3.1 Introduction 3.2 Principles and procedures 3.3 Applications (FYI) TEXTBOOK REFERENCES 1. Biology, Campbell and Reece, 9th Edition, pgs 442 - 469 2. Biology of Microorganisms, Brocks, Chapter 8 Genetic Engineering and Biotechnology 3. Genetics, Analysis & Principles, Robert J.Brooker, 2nd Edition, Chapter 20 Structural Genomics INTERNET ANIMATIONS Polymerase Chain Reaction http://www.dnalc.org/view/15475-The-cycles- of-the-polymerase-chain-reaction-PCR-3D- animation- with-no-audio.html http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html# http://www.sumanasinc.com/webcontent/animations/content/pcr.html http://learn.genetics.utah.edu/content/labs/pcr/ Gel Electrophoresis http://www.dnalc.org/resources/animations/gel electrophoresis.html http://learn.genetics.utah.edu/content/labs/gel/ http://www.dnalc.org/resources/animations/gelelectrophoresis.html Southern Blotting and Nucleic acid hybridisation http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

Page 4 of 27 0. Overview of Molecular Techniques for DNA Analysis A target DNA sequence can refer to either a gene of interest or non-coding sequences. What are you trying to do? Possible Methods 1. Extract total DNA (Breaking cells apart to remove DNA from the other cellular content) 2. Isolate target DNA sequence(s) 3. Separation of target DNA sequence/s from other DNA fragments (according to molecular size) 4. Visualization of DNA

Page 5 of 27 1. Polymerase chain reaction (PCR) 1.1 Introduction The molecular biologist studying a particular gene faces a challenge. Naturally occurring DNA molecules are very long and a single molecule carries many genes. Moreover, in many eukaryotic genomes, genes only occupy a small portion of the chromosomal DNA, the rest being noncoding nucleotide sequences. Hence to work directly with specific genes or other DNA segments, scientists have developed laboratory techniques to obtain multiple identical copies of a specific DNA sequence, a process known as DNA cloning. The production of multiple copies of a single gene is called gene cloning. DNA cloning can be achieved using cells (e.g. E.coli bacteria) or through polymerase chain reaction (PCR) using a thermal cycler. DNA cloning using cells remains the best method for preparing large quantities of a particular gene or DNA sequence. However, when DNA sample for cloning is in small quantities or impure, PCR is a q uicker and more selective method by which large quantities of a specific DNA sequence can be obtained in a short time. Devised in 1985 by Dr. Kary Mullis, PCR revolutionized DNA research. For inventing this method, Dr. Mullis was award the 1993 Nobel Prize in Chemistry. Polymerase chain reaction (PCR) ➢ Polymerase chain reaction (PCR) is a technique in which a sequence of DNA is amplified (copied many times) in vitro (outside of living organism) using a thermal cycler. ➢ In this method, any specific DNA target segment within one or many DNA molecules can be quickly amplified in a test tube. With automation, PCR can make billions of copies of a target segment of DNA in only a few hours (e.g. 2-3 hours). 1.2 Principles and procedures (a) Reagents and equipment for PCR What is needed? Description 1. DNA sample (source DNA) to be amplified • The DNA to be amplified can be obtained from a small original sample from any source . (eg. DNA extracted from blood, sperm, or any other tissue, from older forensic specimens, bacterial colonies or purified DNA). • The target DNA sequence to be amplified by PCR must be known to some extent so that D