Part I - Cloning In General

Why Clone? Information can be obtained that will aid in: A. Gene therapy: Used to treat certain genetic conditions. http://www.genetherapynet.com/literature.html B. Genetic engineering of organisms: To introduce a characteristic of value into an organism. http://online.liebertpub.com/loi/GEN

Steps in Cloning 1. Isolate the DNA from the organism that contains the desired gene. 2. Cut the isolated DNA into fragments with a restriction enzyme. This produces staggered cuts at the ends. 3. Cut a plasmid vector with the same restrictive enzyme to form staggered ends that correspond to the ones on the DNA fragments. 4. Mix the DNA and plasmid together so that the fragments will become inserted into the plasmid. 5. Incorporate plasmids into bacterial host cells by transformation. 6. Plate the cells out on an agar medium. 7. Identify and isolate the colonies containing the desired cloned gene.

Cloning Animations www.highered.mcgraw- hill.com/sites/0072556781/student_view0/chapter14/anim ation_quiz_1.html http://www.sumanasinc.com/webcontent/animations/cont ent/plasmidcloning.html

Isolate DNA http://www.teachersdomai n.org/resource/biot09.biot ech.tools.extraction/

Restrictive Enzyme: Prepare pBluescript II and DNA strands 1. BamHI is the restrictive enzyme used to cut the strands and plasmid. 2. “Sticky ends” will result that will covalently bond together. 3. DNA ligase will seal the ends by creating phosphodiester bonds.

Plasmid Vector Features An ori sequence A selectable marker 1 or more unique restriction enzyme cleavage sites Origin of DNA replication needed for plasmid to replicate in E. coli. Gene that codes for a characteristic that can be used to indicate if a cell does or does not contain the cloning vector – typically resistance to ampicillin. 18 restriction sites.

Example of Plasmid Vector: pBluescript II Good choice because it has a: 1. very active ori resulting In a high copy number. 2. the amp selectable marker for ampicillin resistance. 3. 18 restriction sites.

Transforming Methods Chemical transformation: Electroporation: The bacteria is treated chemically with CaCl2 which allows taking up of the plasmids because the Ca2+ binds with negative molecules, allowing the plasmids to slip through the membrane of the bacteria Electroporation: Electric shock is delivered to the cells, causing temporary disruption of the cell membranes allowing the DNA to enter.

Transformation Animation http://dnalc.org/resources/animations/transformation2.ht ml

Part II: Example of a Gene of Interest to Be Studied HBB gene – codes for mutation of beta hemoglobin strand that results in Sickle Cell Anemia

Model of a normal hemoglobin molecule Hemoglobin molecules contain 2 alpha and 2 beta polypeptide strands. In sickle cell anemia, the gene that codes for the beta strands has mutated resulting in an abnormal beta strand and hemoglobin molecule. http://susankannasjackiegunderson .wikispaces.com/

Hemoglobin Genetics The gene that codes for the beta chain of the hemoglobin molecule is on chromosome 11. Genes are made up of nucleotides. Every 3 nucleotides code for an amino acid. In sickle cell anemia the 6th amino acid glutamic acid (GAG) is replaced with a valine (GUG). The result of this mutation is that the structure of the beta chain is altered so that there is a nick in it that the other beta chain is attracted to when it shouldn’t be. Structure is altered and so is the function.

Disease that causes the r. b. c Disease that causes the r.b.c. to sickle and get stuck in the blood vessel. Blood vessels are damaged. Blood flow is disrupted and this results in less oxygen to the body tissues. Sickle Cell Anemia http://www.ncbi.nlm.nih.gov/pub medhealth/PMH0001554/

Part III – Cloning HBB gene

1. Use an Expression Vector Additional Features: 1. Promoter upstream of multiple cloning site. 2. Transcription terminator down- stream of the multiple cloning site. 3. DNA sequence encoding the Shine-Dalgarno sequence for translation initiation located between the promoter and the multiple cloning site. 1. Use an Expression Vector

2. Insert the Gene of Interest Cut the strand and vector with restrictive enzyme BamHI. 2. Insert the Gene of Interest

3. Transform the plasmid into E. coli and translate the cDNA strand.

4. Use an Antibody Probe to Screen Specific Gene of Interest E coli colonies are grown on selective medium. Individual colonies are transferred to a filter and the protein coded for is expressed. The expressed proteins are reacted with a radioactive antibody that binds to the protein encoded for by the gene of interest.

5. Identify with an Autoradiogram After the unbound antibody is washed off, the colony that expressed the gene of interest could be identified with an autoradiogram.

Bibliography www.highered.mcgraw- hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_1.html http://www.sumanasinc.com/webcontent/animations/content/plasmidcloning.html http://www.teachersdomain.org/resource/biot09.biotech.tools.extraction http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001554/ http://dnalc.org/resources/animations/transformation2.html http://susankannasjackiegunderson.wikispaces.com/ Russell, P., (2010). iGenetics. San Francisco, CA: Pearson Benjamin Cummings.