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Working with DNA: Isolation and Fingerprinting Funding and support received from… Today’s Agenda: 1) Introduction 2) Safety 3) Basic Practice “Using a Pipetteman” 4) DNA Isolation Procedures 5) Restriction Enzymes and Gels 6) Yellowstone National Park and Bacterial Mats 7) Practice DNA Fingerprinting Problems 8) Analysis of our Fingerprinting Gels 9) Bacteria and DNA Basics 10)Closing Our Research Project - What We Are Cloning and Why • We hope to identify • new hot spring bacteria that cannot be grown on lab media To study these organisms, we extract DNA from hot springs that contain unknown bacteria Our Research Project - What We Are Cloning and Why • We clone a specific • • identification gene (the 16S gene) from the hot spring DNA We place each hot spring gene into E. coli, our cloning factory And then we fingerprint and DNA sequence each hot spring clone Words to the cautious… Neither the E. coli we use nor the hot spring bacteria we study have ever been shown to be pathogenic. Although you will be working with E. coli, you will never come in contact with hot spring bacteria… just their DNA after it has been extracted from the once-living cells. Introduction: • All living things contain cells • Eukaryotes: more than one cell • Prokaryotes: one cell organisms The Boring (Yawn!!) Eukaryotic Plant and Animal Cells… The Exciting Bacterial Cell… Bacteria come in many different shapes and sizes…take a quick look… Bacteria can replicate easily… • To grow, bacteria divide and divide and divide again. • Problem: If you started with only 1 bacterial cell, and it divided 10 times, how many bacteria would you then have?? Bacteria are everywhere… Don’t panic!! This is a good thing. We have bacteria growing on our bodies which are supposed to be there. What are Bacteria? • Bacteria are prokaryotes, meaning they are only one celled organisms. They are very small and can be harmful or beneficial. Bacteria can cause diseases, like we all know… Bacteria can also have beneficial uses… Bacterial Cell Components… Plasmids can also be found in bacterial cells: • Plasmids are: Mini• • • • chromosomes found only in some bacteria (1,000-10,000 base pairs) Free-floating in the cytoplasm - not membrane-bound like chromosome Naturally carry many antibiotic resistance genes Replicate on their own Plasmids and Cloning • Bacteria are used in genetic engineering and cloning because they serve as the factories for expressing foreign genes like insulin. Without plasmids, there would be no way to clone and express foreign genes. Now we are going to do some work!!! DNA Precipitation What are we using now? • 3M Sodium acetate: contributes ions to bind with positive phosphates open on DNA • Isopropanol: polar solution which attaches to DNA for precipitation • - 80C Freezer: Speeds the precipitation reaction with low amounts of DNA DNA… the code of life What do we know about DNA? • Structure: Composed of nucleotides (monomer) consisting of: 1) phosphate group 2) deoxyribose sugar 3) one of four nitrogen bases What do we know about DNA? • Structure: Nitrogen bases are named: - adenine (A) - guanine (G) - thymine (T) - cytosine (C) What do we know about DNA? • Structure: • The structure of these nucleotides determines how they fit together. • Adenine fits with Thymine • Guanine fits with Cytosine What do we know about DNA? • Structure: • DNA is “double-stranded” • The nucleotides are • • linked together covalently Phosphate – Sugar – Phosphate – Sugar etc. This is the “backbone” What do we know about DNA? • Structure: • The two strands are oriented in opposite directions • The two strands are wound around each other forming the “helix” structure What do we know about DNA? • Function: • Codes for 80,000 genes, which form proteins…the building blocks of life. Eukaryotic Deoxyribonucleic Acid • DNA for Short • Double helix - two strands made up of A, T, G, and C bases • Complex organisms - many linear chromosomes (10,000,000,000 or more base pairs) Plant or Animal DNA Strand: Prokaryotic Deoxyribonucleic Acid • Bacteria - one circular chromosome (1,000,000 base pairs) • Chromosomes, in both cases, are held by proteins to the cell or nuclear membrane • Most RNA is translated into proteins that have structural or functional jobs in cells Bacterial DNA Strand Let’s get our samples now and continue on with our isolation… • Centrifuge: Spins solution at high speed to concentrate DNA at the bottom • TE: buffer at pH 8.0 • RNAse: enzyme which removes RNA present in sample through digestion Restriction Enzymes and Gels Restriction Enzymes • Cut specific sequences of DNA • Many different kinds • Named after organism they came from, enzyme number • E.g. EcoR1 Bacteria Produce Restriction Enzymes • Uniquely bacterial protection mechanism…why? • Restriction enzymes are short nucleotide sequences isolated from bacteria cells that protect them from virus. Bacteria Produce Restriction Enzymes • When a viral DNA enters the bacterial cell, the restriction enzyme is able to recognize a specific sequence (restriction site) on the DNA molecule, which is usually 4-8 nucleotides long. The restriction enzyme will cut the viral DNA at these sites and hence restrict the growth of the virus. Bacteria Produce Restriction Enzymes • Several hundreds of these enzymes have been isolated from various organisms and most are available commercially. These enzymes are used to cut a segment of gene from a human DNA molecule. DNA Fingerprinting DNA Fingerprinting • DNA fragments are separated using gel electrophoresis • Each band represents the DNA which has been cut into smaller pieces using restriction enzymes Gel Electrophoresis From your studies of DNA, can you tell me what charge DNA has? Gel Electrophoresis • Gel is made of water and agarose • Wells on one end are where gels will be loaded with our samples Gel Electrophoresis • The gel box contains water and buffer to keep the pH constant • Gel box has platinum wire that conducts protons and electrons • Gel box will be wired to the power source following the load Gel Electrophoresis • To the strand of DNA moving through the agarose, the gel looks like a big mesh-like maze • The DNA travels through the maze as fast as it’s size will allow Gel Electrophoresis • DNA moves from the negative towards the positive • Smaller – faster • Larger – slower • Where will these three end? Gel Electrophoresis • Review: ** DNA travels – to + ** When the power supply turns off, we can see where the bands are and infer which are bigger and smaller ** Small goes far ** Large goes not far DNA Fingerprinting Questions and Answers Do you know the answers to these questions? DNA Fingerprinting • How good (accurate) is it at identification. For example, is it as good as classical fingerprints? Question 1: • How good (accurate) is it at identification. For example, is it as good as classical fingerprints? • Answer: In theory, with the exception of identical twins, EVERYONE on this planet has a different DNA fingerprint. That is, DNA fingerprinting IS as good (distinctive/unique/specific) as classical fingerprinting for identification. Question 2: • What are its advantages? • Answer: In theory DNA fingerprinting will work with much smaller amounts of material than a classical fingerprint & DNA lasts much longer than classical fingerprints. DNA-containing samples that are many years old (up to 25 million yr.) are still usable. Only very tiny quantities of DNA are required in order to carry out a highly accurate test. For example, dried blood, semen, spit, skin etc. on samples stored in dusty files for years are still usable. Samples of mixed DNA's can also be used. DNA containing evidence is much harder to clean up at a crime scene than other evidence, like classical fingerprints. Question 3: • What are its limitations? • Answer: There currently are no accepted Federal standards for controlling the quality of DNA testing nationwide. Poor quality & poorly controlled testing can lead to QUESTIONABLE and SHODDY RESULTS. • Even if there is a perfect match between DNA, you can not say HOW the DNA containing sample got there or WHEN. In the O.J. trial a VALID question was raised about the possibility of evidence being planted. What makes this charge so powerful is the EXTREME SENSITIVITY of the procedure. Now, how do we come up with those different bands? Answer: Restriction Enzymes Let’s do an example of DNA Fingerprinting together… DNA Fingerprinting Example: • Two men fitting the description of a robber were caught in the vicinity of the crime. Both had cuts on their arms which they “explained away.” DNA samples were taken from each suspect and from the broken window at the scene of the crime. DNA Fingerprinting Example: • Using DNA Fingerprinting and Restriction Enzymes, we can determine which of the men was the robber!! • We can cut each sample (one from each suspect and one from the crime scene) with two different enzymes, run them on a gel and compare the results So, how do we organize what we know? We organize the gel lanes… Lane Description: 1 DNA sample from crime scene cut w/ Enzyme 1 2 DNA sample from crime scene cut w/ Enzyme 2 3 DNA sample from Suspect 1 cut with Enzyme 1 4 DNA sample from Suspect 1 cut with Enzyme 2 5 DNA sample from Suspect 2 cut with Enzyme 1 6 DNA sample from Suspect 2 cut with Enzyme 2 Fingerprinting Gel Projected Results: Practical applications of DNA technology Practical applications of DNA technology • Diagnosis of diseases includes: – – – – Huntington’s PKU cystic fibrosis Duchenne’s muscular dystrophy Practical applications of DNA technology • Human gene therapy • Somatic cell therapy versus germ cell therapy Practical applications of DNA technology • Pharmaceutical products: Insulin human growth hormone • Protection from viral infection Practical applications of DNA technology Forensic uses DNA fingerprinting RFLPs and simple tandem repeats (microsatellite DNA repeats of different lengths) Practical applications of DNA technology • Environmental uses: • Genetically engineered microbes for mining, cleaning up toxic wastes, etc. Practical applications of DNA technology Agricultural uses • Animal husbandry – Transgenic animals – Gene knock-in or knockout animals (requires homologous recombination) – Cloned animals • Genetic engineering in plants – Can grow many plants from a single cell