Bio 3A Lab: DNA Isolation and PCR 1 Page 1 of 7 Bio 3A Lab: DNA Isolation and the Polymerase Chain Reaction Objectives • Understand the process of DNA isolation • Perform DNA isolation using cheek cells • Use thermal cycler and Taq polymerase to perform DNA amplification • Gain an understanding of the PCR process Introduction You are about to perform a procedure known as the polymerase chain reaction (PCR). This procedure will be used to amplify a specific sequence of your own DNA in a test tube. This particular piece of DNA is present in the genes of many but not all people. Analysis of the data generated in this laboratory will enable you to determine whether your genomic DNA carries this piece of DNA, or not. The genome, composed of DNA, is our hereditary code. This is the so-called "hard-wiring", the blueprint that controls much of why we look like we do, why we act like we do, and how we do the things we do. Molecular biology is the study of genes and the molecular details that regulate the flow of genetic information from DNA, to RNA and proteins, from generation to generation. Biotechnology uses this knowledge to manipulate organisms’ (microbes, plants or animals) DNA to help solve human problems. Within the molecular framework of biology, DNA, RNA and proteins are closely tied to each other. Because proteins and enzymes ultimately play such a critical role in the life process, scientists have spent many lifetimes studying proteins in an attempt to understand how they work. With this understanding it was believed we could cure, prevent and overcome disease and physical handicaps as well as explain exactly how and why organisms exist, propagate and die. However, the complete answer to how and why does not lie solely in the knowledge of how enzymes function, we must learn how they are made. If each enzyme is different, then what controls these differences and what is the blueprint for this difference? That answer lies within our genome, or genetic code. Thus, you may realize why researchers today, in an attempt to understand the mechanisms behind the various biological processes, study nucleic acids as well as proteins in order to get a complete picture. In the last 20 years, many advances in the areas of nucleic acid techniques have allowed researchers the means to study the roles that nucleic acids play in biology. It took the imagination and hard work of many scientists to reveal the answers to one of the most mysterious puzzles of life - understanding the mechanisms that control how DNA is translated into proteins within living cells. Before Beginning this Lab, See If You Can Answer the Following Questions • How is DNA faithfully passed on from generation to generation? • What causes genetic diseases in some people but not others? • How do scientists obtain DNA to study? • What secrets can DNA tell us about our origins? • What human problems can an understanding of DNA help us solve? • Should we unlock the secrets held in this most basic building block of life? PCR Set the Stage for a Scientific Revolution In 1983, Kary Mullis at Cetus Corporation developed the molecular biology technique that has since revolutionized genetic research. This technique, termed the polymerase chain reaction (PCR), transformed molecular biology into a multidisciplinary research field within 5 years of its invention. Before PCR, the molecular biology techniques used to study DNA required such a high level of expertise that relatively few scientists could use them.Bio 3A Lab: DNA Isolation and PCR 1 Page 2 of 7 The objective of PCR is to produce a large amount of DNA in a test tube (in vitro), starting from only a trace amount. Technically speaking, this means the controlled enzymatic amplification of a DNA sequence, or gene, of interest. The template strands can be any form of double-stranded DNA such as genomic DNA. A researcher can take trace amounts of genomic DNA from a drop of blood, a single hair follicle or cheek cell (in theory, only a single template strand is needed to copy and generate millions of new identical DNA molecules) and make enough to study. Prior to PCR, this would have been impossible. It is the ability to amplify the precise sequence of DNA of interest that is the true power of PCR. PCR has made an impact on four main areas of genetic research: gene mapping, cloning, DNA sequencing and gene detection. PCR is now used as a medical diagnostic tool to detect specific mutations that may cause genetic disease, is used in criminal investigations and courts of law to identify suspects on the molecular level, and has been a powerful tool in the sequencing of the human genome. Prior to PCR the use of molecular biology techniques for therapeutic, forensic, pharmaceutical, agricultural or medical diagnostic purposes was not practical nor cost effective. The development of PCR technology transformed molecular biology from a difficult science to one of the most accessible and widely used disciplines of biotechnology. Procedure 1: DNA Extraction and Template Preparation To obtain DNA for use in the polymerase chain reaction you will extract the DNA from your own living cells. It is interesting to note that DNA can be also extracted from mummies and fossilized dinosaur bones. In this lab activity, you will be isolating DNA from epithelial cells that line the inside of your cheek. This is accomplished by using a sterile pipet tip to gently scrape the inside of both your cheeks about 10 times each to scoop up the cells lining the surface. You will then boil the cells to rupture them and release the DNA they contain. To obtain pure DNA for PCR you will use the following procedure. The cheek cells in the pipet tip are transferred into a micro test tube containing 200 µl of InstaGene matrix. This particulate matrix is made up of negatively charged microscopic beads that "chelate", or grab metal ions out of solution. It acts to trap metal ions, such as Mg 2+, which are required as catalysts or cofactors in enzymatic reactions. Your cheek cells will then be lysed or ruptured by heating to release all of their cellular constituents, including enzymes that were once contained in the cheek cell lysosomes. Lysosomes are sacs within the cells cytoplasm that contain powerful enzymes, such as DNAases, which are used by cells to digest the DNA of invading viruses. When you rupture the cells, these DNAases can digest the released DNA of