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CHAPTER 20: DNA TECHNOLOGY AND GENOMICS I. II. INTRODUCTION A. Recombinant DNA 1. Definition: B. Genetic Engineering 1. Definition: C. Biotechnology 1. Definition: DNA CLONING A. Introduction 1. Why is it so difficult to work with the just a gene? B. Page 1 of 25 DNA technology makes it possible to clone genes for basic research and commercial applications: an overview. 1. Be able to describe the role of plasmids in the cloning of genes 1 493727018 2. C. Page 2 of 25 Potential uses of cloned genes Restriction enzymes are used to make recombinant DNA 1. Restriction Enzymes or Endonucleases 2 493727018 D. Page 3 of 25 2. Restriction Sites and Restriction fragments 3. Sticky Ends Genes can be cloned in recombinant DNA vectors: a closer look 1. Cloning Vector a) This is simply the tool that will carry the gene of interest. b) It is usually DNA that will carry the new or foreign gene into whatever cell we want the gene to be expressed. c) Plasmids can be vectors d) Bacteria are common host cells that will accept the plasmid, reproduce the plasmid, and therefore it has cloned the gene. 3 493727018 2. Procedure for Cloning a Eukaryotic Gene in a Bacterial Plasmid a) Isolation of vector and gene-source DNA b) Page 4 of 25 Insertion of DNA into the vector 4 493727018 Page 5 of 25 c) Introduction of the cloning vector into cells d) Cloning of cells (and foreign genes) 5 493727018 e) 3. Identification of cell clones carrying the gene of interest (i) We can detect the gene or DNA by exposing the double stranded DNA to chemicals or heat to denature the DNA and then expose the single stranded DNA to a radioactive probe of DNA that pairs up with the gene of interest (Nucleic Acid Hybridization) (ii) Sometimes this probe can have a fluorescent tag or marker on it, like the GFP that we have spoken about or will speak about. (iii) So what we have done then is to “screen” for the gene of interest using a nucleic acid probe. (iv) Once the bacteria that have the gene of interest we will want to grow them in large quantities to isolate the gene product- the protein. Cloning and Expressing Eukaryotic Genes: Problems and Solutions a) Expression Vector: used to overcome some of the difficulties of putting a eukaryotic gene into a prokaryote. Page 6 of 25 6 493727018 b) Presence of noncoding regions c) YACs or how to avoid those prokaryotic problems Page 7 of 25 7 493727018 d) Protein Modifications cannot be done by bacteria e) Eukaryotic cells just cannot take up the DNA like bacteria so we Electroporate them!! Page 8 of 25 8 493727018 E. E. Cloned Genes are stored in DNA libraries 1. Genomic Library: when a bunch of cut up DNA is exposed to bacteria, all of the pieces can be taken up by the bacterial plasmids. So then each plasmid has a particular segment of the DNA that was all cut up. This is a library of the DNA. Some of these plasmids will be valuable, others won’t. 2. Viruses can also be used to make a library: pieces of foreign DNA can be inserted into the virus’s genome using a restriction enzyme and ligase. This then is packaged in a capsid and allowed to infect cells. So as the virus’ DNA replicates, so does the foreign DNA of interest. 3. cDNA or complementary DNA (sometimes called copy DNA) a) You take all the mRNA that is produced by a cell and expose it to reverse transcriptase to make DNA from all the expressed genes. This then is a cDNA Library but it represents only those genes expressed at the time of the study. b) Liver cells may produce a totally different cDNA library than lung cells. That way we know what different genes are being transcribed by these different types of tissue. The polymerase chain reaction (PCR) clones DNA entirely in vitro (know this; this was part of one of the essays on the AP Bio Exam 2002) Page 9 of 25 9 493727018 Page 10 of 25 10 493727018 III. DNA ANALYSIS AND GENOMICS A. Page 11 of 25 Introduction 1. What are some of the questions that can be asked (by Dr. Campbell as well as the AP Committee) once we have large numbers of identical segments of DNA? 2. What is Genomics? 4. The “other” method to compare samples of DNA –Gel Electrophoresis. 11 493727018 B. Restriction fragment analysis detects DNA differences that affect restriction sites. 1. Page 12 of 25 Be able to describe this method for an essay question. More detail should come from a lab we do. You are responsible for a detailed description of this process. Figure 20.8 and 20.9 12 493727018 2. Nucleic Acid Hybridization (pg 385 and Fig 20.10) a) Know the procedure and understand RFLPs( RFLPs was part of the 2002 AP Exam). Page 13 of 25 13 493727018 C. Entire Genomes can be mapped at the DNA Level 1. Introduction and the Human Genome Project 2. Genetic (Linkage) Mapping a) Genetic mapping is finding out where “genes” are but what we many times are locating are markers for these genes. b) When we know the location of various genes we can determine if they are linked based on their distances between each other. 3. Page 14 of 25 Physical Mapping: Ordering DNA Fragments 14 493727018 Page 15 of 25 4. DNA Sequencing 5. Alternative Approaches to Whole-Genome Sequencing 15 493727018 D. Genome sequences provide clues to important biological questions 1. Introduction 2. Analyzing DNA Sequences a) b) Page 16 of 25 Introduction (i) Stored in data banks (ii) Researchers can scan these sequences to (iii) see if a particular gene is similar between organisms (iv) promoters are similar (v) splicing sites are similar (vi) Sequences that are found to be similar are called Expressed Sequence Tags (ESTs) Surprisingly Few Genes in the Human Genome 16 493727018 Page 17 of 25 c) Studying and Comparing Genes 3. Studying Gene Expression 17 493727018 4. Determining Gene Function a) Page 18 of 25 Disable the gene or mutate it and see what effect it has on the organism. 18 493727018 5. Page 19 of 25 b) In vitro mutagenesis: In vitro means to be done outside of the cell or organism so here a gene is removed so it is outside of the cell and is altered. The altered form is then reinserted into the cell and its expression is observed. Perhaps a protein is deformed or a certain tissue doesn’t form in an embryo so this suggests the gene has something to do with controlling cell differentiation. c) RNA interference (RNAi): well, I’ve never heard of this before so. . . (i) Purpose: used to shut off the expression of a specific gene. (ii) Take some dsRNA. This dsRNA has a sequence that matches a gene that will cause the breakdown of the mRNA that belongs to the gene you are interested in. (iii) So you have no polypeptide made from the mRNA, hence no function and you can observe the effects. Future Directions in Genomics a) Proteomics b) Bioinformatics 19 493727018 IV. Practical Applications of DNA Technology A. DNA technology is reshaping medicine and the pharmaceutical industry 1. Introduction a) identifying genes of disease and health is part of DNA Tech b) finding mutations is part of DNA Tech c) understanding how genes are expressed and their control mechanisms helps you to understand how disease occurs. d) Different arrangement of gene expression (microarrays) help you to understand a larger picture of many genes and their expression related to disease and health 2. Diagnosis of Diseases 3. Page 20 of 25 Human Gene Therapy 20 493727018 4. Page 21 of 25 Pharmaceutical Products 21 493727018 B. Page 22 of 25 DNA technology offers forensic, environmental, and agricultural applications 1. Forensic Uses of DNA Technology 22 493727018 Page 23 of 25 2. Environmental Uses of DNA Technology a) Bacteria decomposing oil b) Bacteria extracting heavy metals from contaminated soil c) Bacteria degrade organic pollutants 3. Agricultural Uses of DNA Technology a) Animal Husbandry and “Pharm” Animals 23 493727018 b) Page 24 of 25 Genetic Engineering in Plants 24 493727018 C. Page 25 of 25 DNA technology raises important safety and ethical questions 25 493727018