Chapter 4: Introduction to Studying DNA Introduction to Biotechnology, BIOL1414 Austin Community College, Biotechnology Dept
Learning Outcomes • Describe the structure and function of DNA and explain the process by which it encodes for proteins • Describe how DNA is replicated in the cell • Differentiate between eukaryotic and prokaryotic chromosomal structure and explain how this difference impacts gene regulation in the two cell types • Describe the process of gel electrophoresis and explain how the characteristics of molecules affect their migration through a gel Note about this PowerPoint – There are several links in this PPT that allow you to explore more into different topics. Some of these links are animations, movies, or exercises. Please note, you must be in the slide show to activate the links. You can press F5 any time to active the slide show and “Esc” to exit.
DNA Structure & Function • The manipulation of genetic information, DNA and RNA codes, is at the center of most biotechnology research and development. http://www.historyforkids.org/scienceforkids/biology/cells/dna.htm
DNA Discovery (visit DNAi.org) • Miescher – identified a nuclear substance he called nuclein • Griffith – performed the first transformation • Avery, McCarty, and Macleod – identified Griffith’s transforming factor as DNA • Chargaff – proved that the percentage of the DNA bases adenine always equaled thymine and guanine always equaled cytosine • Wilkins, Franklin, Watson & Crick – demonstrated the structure of DNA
The Central Dogma of Biology. Proteins are produced when genes on a DNA molecule are transcribed into mRNA, and mRNA is translated into the protein code. This is called “gene expression.” At any given moment, only a relatively small amount of DNA in a cell is being expressed.
Similarities in DNA Molecules Among Organisms • Virtually all DNA molecules form a double helix • The amount of adenosine equals the amount of thymine • The amount of guanosine equals the amount of cytosine • Nucleotides in each strand are oriented in the opposite direction of the other strand • Nitrogenous bases • DNA undergoes semi-conservative replication
Variations in DNA Molecules • DNA from organism to organism varies in: • The number of DNA strands in the cells of an organism • The length in the base pairs of the DNA strands • The number and type of genes and non-coding regions • The shape of the DNA strands (circular vs linear)
DNA Structure • The nucleotides in one chain of the helix face one direction, while those in the other strand face the other direction : ANTI-PARALLEL • Each nucleotide contains a sugar molecule, a phosphate group, and a nitrogenous base. • Nitrogenous bases from each strand bond to each other in the center through Hydrogen-bonds.
Structure of DNA - Nucleotides • Deoxyribose Sugar • Phosphate • Nitrogen Base
Structure of DNA - Nucleotides • Purines – double ring • Pyrimidines – single ring
Structure of DNA Nucleic Acid Overview
What is a Gene? • A gene is a sequence of nucleotides that provides cells with the instructions to synthesize a specific protein. • Note, that not all genes produce protein! • Most genes are 1000-4,000 nt long and encode for a particular trait • Note, some traits are encoded by one gene, but most are determined by multiple genes!
What is a genome? • DNA contains the instructions for life – genes • All the DNA in an organism’s cells is called a genome • The humangenomecontains over 3 billion bases and 23,000 genes. • The study of genomes is called genomics • Genomics is an exciting career area!
DNA Replication • When DNA makes an exact copy of itself • Growth & Development • Replace aging/damaged cells DNA Replication Animation
DNA Replication • DNA replicates in a semi-conservative fashion in which one strand unzips and each side is copied. • It is considered semi-conservative since one copy of each parent strand is conserved in the next generation of DNA molecules.
DNA Replication • The first step in DNA replication is for the enzyme, helicase, to unzip the double stranded DNA molecule.
DNA Replication Proteins hold the two strands apart. An RNA primer lays down on each strand of DNA.
DNA Replication • DNA polymerase extends the primer by adding complementary nucleotides. • DNA polymerase can only extend in the 5’ → 3’ direction
DNA Replication Leading strandfollows helicase. Lagging strandmust wait for replication fork to open and therefore forms discontinuous Okazaki fragments. Ligase seals the nicks in the DNA backbone between the Okazaki fragments. helicase
Let’s put it all together • Click on the animation below. • Select the button for the “whole picture”. DNA Replication Animation
Sources of DNA • In nature, DNA is made in cells. Mammalian Cell Culture • Growing mammalian cells in culture is more challenging than growing bacterial cells • Mammalian cells are grown in a broth culture Viral DNA • Viruses are classified according to the type of cell they attack: • Bacterial (bacteriophages) • Plant • Animal
Prokaryotic DNA • Gene Expression in prokaryotes is much more simple than eukaryotes • An operon contains the controlling elements that turn genetic expression ON and OFF.
Let’s put it all together • Click on the animation below. Animation of lac operon Video of lac operon
Bacterial Cell Culture • Some bacteria grow well in liquid medium – broth • Some bacteria prefer solid medium – agar • Some grow on both for different purposes http://cals.arizona.edu/main/spotlight/how-microbes-take-out-trash http://www.sciencelearn.org.nz/Contexts/Enviro-imprints/Sci-Media/Images/E.coli
Eukaryotic DNA • Eukaryotic genes have a promoter to which RNA polymerase binds, but they do not have an operator region. • Transcription factors may bind at enhancer regions and increase gene expression.
Mammalian Cell Culture • Growing mammalian cell culture is far more challenging and expensive than bacterial cell culture • Typically grown in broth culture in special flasks • Specific media designed to have all special nutrients of that cell type • Special indicators can be added to monitor grown (such as phenol red)
Isolating and Manipulating DNA • Identification of molecules for our benefit – Insulin for example • Isolation of DNA (gene) • Manipulation of DNA – insert into a different organism to produce the gene product • Harvest to the molecule of interest from the host organism
Using Gel Electrophoresis to Study Biological Molecules Gel Electrophoresis: • Most commonly used when separating pieces of DNA no smaller than 50 bp and no larger than 25,000 bp • The gel is “run” until molecules of different sizes are thought to have completely separated. Components of Gel Electrophoresis • Powdered agarose • Boiling buffer solution • Running buffer • DNA stain • Sample load buffer
Gel Electrophoresis • Electrophoresis is a molecular technique that can (depending on application) separate nucleic acids and proteins based on: Size and/or +-+Charge+-+ Shape
DNA Agarose Gel Electrophoresis • Click here for animation on Agarose gel electrophoresis! • Click here to watch a video on Agarose gel electrophoresis!
Agarose Gel Electrophoresis • DNA is analyzed by size alone on agarose gel electrophoresis. • DNA is a negatively charged molecule and therefore is attracted to positive charges.
Agarose Gel Electrophoresis • Agarose provides a matrix through which DNA molecules migrate. • Larger molecules move through the matrix slower than small molecules • The higher the concentration of agarose, the better the separation of smaller molecules
Agarose Gel Tray. Gel trays differ depending on the manufacturer. Each has some method of sealing the ends so that liquid agarose can mold into a gel. Some gel trays, such as those made by Owl Separation Systems, make a seal with the box, so casting a gel is simple. Other trays require masking tape on the ends to make a mold. Still others, like the one shown here, have gates that screw into position: up for pouring the gel and down for running the gel.
DNA Agarose Gel Electrophoresis • For the gel box to conduct electricity and establish an electric field with a positive end (red wire) and a negative end (black wire), the solution in the gel box must contain ions. • The smallest molecules run fastest thru the gel
DNA Agarose Gel Electrophoresis • DNA fragments separate according to size. • Smaller fragments run faster through the agarose mesh
Agarose Gel Electrophoresis • How to make an agarose gel: • Weigh out a specified amount of agarose powder. • Add the correct amount of buffer. • Dissolve the agarose by boiling the solution. • Pour the gel in a casting tray. • Wait for the gel to cool and solidify
Agarose Gel Electrophoresis • How to make an agarose gel: • Place gel in chamber and cover with buffer • Add loading dye to the sample • Load sample on to the gel. • Run at constant voltage
Agarose Gel Electrophoresis • How to make an agarose gel: • Stain the gel – (Ethidium Bromide, SYBR green, methylene blue…) • Capture an image of the gel • Analyze results
Genomic DNA Analysis on Agarose Gel • Genomic DNA isolated from Iris Plants . • Not RNase treated. • 1% agarose gel, 100V, 60min M 1 2 3 genomic DNA 10,000 bp 3,000 bp rRNA 1,000 bp
Analyzing RNA • RNA is another very important nucleic acid commonly isolated and analyzed in a biotechnology lab • RNA provides the link between the genetic information stored in DNA and the expression of that information through protein synthesis. • Differences in RNA and DNA structure: • RNA has deoxyribose • RNA has many different 3-D structures • RNA has A, U, G, C nucleotides • RNA is involved in many different functions including transcription, translation and gene regulation to name a few! • Learn More here
Denaturing Agarose Gel - RNA • Unlike DNA, RNA comes in many different 3-D shapes that affect it’s migration through an agarose gel. • If the particle is small and tightly packed it migrates further/faster than a long cylinder shape particle of the same size. • In order to accurately determine and compare sizes of RNA molecules you must first denature the RNA into a linear form. • The equipment is the same as with DNA analysis, but the buffer used is denaturing – either formaldehyde, or glyoxal buffers work well
How to Prepare an Agarose Gel How to set up DNA Agarose Gel Electrophoresis Click here! How to set up RNA Agarose Gel Electrophoresis Click here!
Review Questions Your Turn! Put your name at the top of a sheet of paper, answer these questions and hand in: • Describe the relationship between genes, mRNA, and proteins. • Name the four nitrogen-containing bases found in DNA molecules and identify how they create a base pair. • The strands on a DNA molecule are said to be “anti-parallel.” What does anti-parallel mean? • During cell division, DNA molecules are replicated in a semi-conservative manner. What happens to the original DNA molecule during semi-conservative replication? • How are small strands of DNA separated in a typical biotechnology lab? What equipment is needed?
References • Biotechnology: Science for the New Millennium. 2012. Ellyn Daugherty.