STUDY SHEET Bio 211 Fall 2012 ComGen Exam Chapter 20: DNA Technology 1.

Define and contrast: recombinant DNA, genetic engineering, biotechnology , vector, restriction enzymes 2. Biotechnology and genetic engineering a. Manipulation of genes to produce useful products 3. Recombinant DNA a. DNA formed from different sources 4. Plasmid a. Cloning vector 5. Know the five steps used to insert human genes into bacteria. a. Isolate DNA b. Insertion in vector c. Reinfect cells d. Clone cells e. Identify colonies with recombinant plasmids 6. Describe how radioactive probes are used to identify cloned genes. a. We find the DNA sequence of interest that we want (i.e. the plasmid DNA) , we then create / synthesize a complimentary DNA strand that is labeled with a radioactive isotope, a fluorescent tag, or another molecule so we can track it. b. Nucleic Acid Hybridization 7. What is cDNA? How is it used to express eukaryotic genes? Why is this method needed? a. Following enzymatic degradation of the mRNA, a second DNA strand, comple mentary to the first, is synthesized by DNA polymerase. The resulting double-str anded DNA is called complementary DNA (cDNA). b. DNA produced from mRNA c. To overcome differences in promoters and other DNA control sequences, sc ientists usually employ an expression vector, a cloning vector that contains a h ighly active bacterial promoter just upstream of a restriction site where the eu karyotic gene can be inserted in the correct reading frame. The bacterial host c ell will recognize the promoter and proceed to express\ the foreign gene now lin ked to that promoter. Such expression vectors allow the synthesis of many eukary otic proteins in bacterial cells d. Getting a cloned eukaryotic gene to function in bacterial host cells can be difficult because certain aspects of gene expression are different in eukary otes and bacteria. 8. What are DNA libraries? a. The complete set of plasmid-containing cell clones, each carrying copies of a particular segment from the initial genome, is referred to as a genomic li brary. Each plasmid clone in the library is like a book containing specific inform ation. 9. Describe the 2 different types of vectors commonly used in recombinant D NA techniques. Vector" is an agent that can carry a DNA fragment into a host cell. If it is use d for reproducing the DNA fragment, it is called a "cloning vector". If it is us ed for expressing certain gene in the DNA fragment, it is called an "expression vector". Commonly used vectors include plasmid, Lambda phage, cosmid and yeast artificial chromosome (YAC) Molecular biologists can avoid eukaryotic-bacterial incompatibility by using euk aryotic cells such as yeasts, rather than bacteria, as hosts for cloning and/or expressing eukaryotic genes of interest. Yeasts, single-celled fungi, offer two advantages: They are as easy to grow as bacteria, and they have plasmids, a rari

ty among eukaryotes. Another type of vector widely used in library construction is a bacterial artifi cial chromosome (BAC). In spite of the name, these are simply large plasmids, tr immed down so they contain just the genes necessary to ensure replication. An ad vantage of using BACs as vectors is that while a standard plasmid can carry a DN A insert no larger than 10,000 base pairs (10 kb), a BAC can carry an insert of 100300 kb 10. What is PCR and what is it used for? a. In this technique, any specific target segment within one or many DNA mo lecules can be quickly amplified in a test tube. With automation, PCR can make b illions of copies of a target segment of DNA in a few hours, significantly faste r than the days it would take to obtain the same number of copies by screening a DNA library for a clone with the desired gene and letting it replicate within h ost cells. b. During each cycle, the reaction mixture is heated to denature (separate) the DNA strands and then cooled to allow annealing (hydrogen bonding) of short, single-stranded DNA primers complementary to sequences on opposite strands at e ach end of the target sequence; finally, a heat-stable DNA polymerase extends th e primers in the 5_ 3_ direction. c. The key to automating PCR was the discovery of an unusual heat-stable DN A polymerase called Taq polymerase, named after the bacterial species from which it was first isolated. 11. Describe gel electrophoresis and what it is used for. a. This technique uses a gel made of a polymer, such as the polysaccharide agarose. The gel acts as a molecular sieve to separate nucleic acids or proteins on the basis of size, electrical charge, and other physical properties. Because nucleic acid molecules carry negative charges on their phosphate groups, they a ll travel toward the positive pole in an electric field. As they move, the thick et of agarose fibers impedes longer molecules more than it does shorter ones, se parating them by length. Thus, gel electrophoresis separates a mixture of linear DNA molecules into bands, each band consisting of many thousands of DNA molecul es of the same length. b. DNA moves from negative (-) to positive (+) c. Larger fragments move slower d. Used in DNA fingerprinting 12. Describe what a restriction fragment analysis is. What are RFLPs? a. In this type of analysis, the DNA fragments produced by restriction enzy me digestion (cutting) of a DNA molecule are separated by gel electrophoresis. b. Restriction fragment analysis can be used to compare two different DNA m oleculesfor example, two alleles of a geneif the nucleotide difference affects a r estriction site. A change in even one base pair of that sequence will prevent a restriction enzyme from cutting there. Variations in DNA sequence among a popula tion are called polymorphisms (from the Greek for many forms), and this particular type of sequence change is called a restriction fragment length polymorphism (R FLP, pronounced Rif-lip). If one allele contains a RFLP, digestion with the enzyme that recognizes the site will produce a different mixture of fragments for each of the two alleles. Each mixture will give its own band pattern in gel electrop horesis c. Detects differences in DNA d. RFLPs i. Inherited differences in non-coding fragments ii. Used in genetic mapping 13. Describe how DNA is sequenced. a. Different fragments of DNA are created using PCR b. The ends of the fragments are dyed c. The fragments are filtered through and shot with a laser and read by the detector what color shows up since the fragments are different sizes. 14. What are DNA microarray assays? What can they be used for?

a. Identification of genes in large samples b. A DNA microarray consists of tiny amounts of a large number of single-st randed DNA fragments representing different genes fixed to a glass slide in a ti ghtly spaced array, or grid (see Figure 20.1). (The microarray is also called a DNA chip by analogy to a computer chip.) Ideally, these fragments represent all the genes of an organism. c. With this method, researchers can test thousands of genes simultaneously to determine which ones are expressed in a particular tissue, under different e nvironmental conditions, in various disease states, or at different developmenta l stages. They can also look for coordinated gene expression. d. Technique i. Isolate mRNA ii. Make cDNA by reverse transcription, using fluorescently labeled nucleoti des iii. Apply the cDNA mixture to a microarray, a microscope slide on which copi es of single-stranded DNA fragments from the organisms genes are fixed, with a di fferent gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray. iv. Rinse off excess cDNA; scan microarray for fluorescence. Each fluorescen t spot (yellow) represents a gene expressed in the tissue sample. 15. What are SNPs and what are they used for? a. Single nucleotide polymorphisms (SNPs) i. Single base-pair variations ii. Can help to identify disease alleles iii. Once a region is found that has a SNP shared by affected but not unaffec ted people, researchers focus on that region and sequence it. In the vast majori ty of cases, the SNP itself does not contribute to the disease, and most SNPs ar e in noncoding regions. Instead, if the SNP and a disease-causing allele are clo se enough, scientists can take advantage of the fact that crossing over between the marker and the gene is very unlikely during gamete formation. 16. Describe in detail organismal cloning in plants and animals. Contrast t otipotent and pluripotent cells. a. Plants i. Adult cells can dedifferentiate 1. Totipotent In plants, at least, mature cells can dedifferentiate and then give rise to all the specialized cell types of the organism. Any cell with this potential is said to be totipotent 1. Can give rise to all cell types b. Cloning plants i. Cross section of carrot root cut out ii. Fragments were cultured in nutrient medium; stirring caused single cells to shear off into the liquid iii. Single cells free in suspension began to divide iv. Embryonic plant developed from a cultured single cell v. Plantlet was cultured on agar medium later it was planted in soil c. Animals i. Adult cells are not totipotent ii. Nuclear transplantation 1. Reproductive cloning d. Cloning animals i. Egg cell donor has the nucleus removed from the egg cell ii. Mammary cell donor cells are cultured. iii. Mammary cells are fused with egg cells iv. The fused cells are grown in culture v. The cell starts to develop into an early embryo vi. Implanted in uterus to a third sheep vii. The embryo is developed e. Stem Cells i. Undifferentiated cells 1. Embryonic pluripotent. ES cells hold more promise than adult stem cells

for most medical applications because ES cells are pluripotent, capable of diffe rentiating into many different cell types. a. Can differentiate into many cell types 1. Adult b. Can produce only a few cell types 17. Contrast embryonic and adult stem cells. a. Embryonic stem cells i. Cells that can generate ALL embryonic cell types 1. Liver cells; nerve cells; blood cells b. Adult stem cells i. Cells that generate a limited number of cell types 1. Blood cells 18. Discuss each of the practical applications of DNA Technology below. a. studying disease - Recognize disease alleles before patient is symptomat ic b. gene therapy - Add good allele to patients cells and reinject cells. Not e ffective yet. i. Insert RNA version of normal allele into retrovirus ii. Let retrovirus infect bone marrow cells that have been removed from the patient and cultured iii. Viral DNA carrying the normal allele inserts into chromosome iv. Inject engineered cells into patient c. Drugs - Human proteins grown in bacteria or animals i. Transgenic or genetically modified organisms (GMOs) ii. Insulin, HGH, clotting proteins, hemoglobin iii. Use bacteria to get foreign genes into plant cells 1. The Ti plasmid is isolated from the bacterium Agrobacterium tumefaciens. The segment of the plasmid that integrates into the genome of host cells is cal led T DNA 2. The foreign gene of interest is inserted into the middle of the T DNA us ing methods mentioned before 3. Recombinant plasmids can be introduced into cultured plant cells by elec troporation. Or plasmids can be returned to agrobacterium, which is then applied as a liquid suspension to the leaves of susceptible plants, infecting them. Onc e a plasmid is taken into a plant cell, its T DNA integrates into the cells chrom osomal DNA d. Forensics - DNA testing, on the other hand, can identify the guilty indi vidual with a high degree of certainty, because the DNA sequence of every person is unique e. Environmental cleanup - the remarkable ability of certain microorganisms to transform chemicals is being exploited for environmental a. Heavy metals to something recoverable i. Lead to lead sulfate ii. Copper to copper sulfate f. Agriculture - Herbicide, pest, frost, drought resistance, add in vitamin s 19. Briefly describe the concerns over genetic engineering. a. How much of your food contains GMOs? i. No Labeling b. Little research done on environmental impact i. Super weeds? c. Social concerns i. Small farmers forced to buy expensive seeds 20. Determining gene function In vitro mutagenesis Knock out a gene and look for the effects RNAi o Engineer a RNA fragment to block the gene o This experimental approach uses synthetic double-stranded RNA molecules matching the sequence of a particular gene to trigger breakdown of the genes mess

enger RNA or to block its translation ComGen labs 1. For each lab conducted know the following: a. What procedures were followed b. What materials were used c. What did each procedure accomplish, why was it important to do d. What was in each of the reagents e. What did each reagent do be specific 2. What is the goal of the ComGen project? 3. What organism are we working on and why? 4. Explain the background covered by Dr. Bangera at the start of the quarte r. 5. What did we start with and what are we trying to find? How do we get fr om start to finish? 6. What results did you get after each procedure followed? 7. Explain how you used electrophoresis and why it was significant. Explai n how you constructed a standard curve and what the point of it was.