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BIO403: Biotechnology

Unit 2: Advanced Nucleic Acid Technologies   This unit will discuss advanced nucleic acid technologies.  For example, methods to visualize DNA and cloning vectors are used in everything from creating gene therapies for disease to cleaning toxic oil spills.  While DNA techniques have been established much longer than RNA methods, RNA methods are exciting new and upcoming fields, especially in ribozymes and RNA interference.  Both have far-reaching implications in biotechnology that could lead to cures for disease and entire new biomedical industries.

Unit 2 Time Advisory
This unit should take you approximately 9.5 hours to complete.

☐    Subunit 2.1: 1.0 hour

☐    Subunit 2.2: 2.0 hours

☐    Subunit 2.3: 1.0 hour

☐    Subunit 2.4: 5.5 hours

☐    Subunit 2.4.1: 0.5 hour

☐    Subunit 2.4.2: 2.0 hours

☐    Subunit 2.4.3: 1.5 hours

☐    Subunit 2.4.4: 1.5 hours

Unit2 Learning Outcomes
Upon successful completion of this unit, the student will be able to:

  • Describe advanced nucleic acid technologies.
  • Compare and contrast nucleic acid amplification techniques and determine the expected outcome.
  • Compare and contrast microRNA and RNAi.
  • Describe RNA techniques.    

2.1 Fluorescence in Situ Hybridization (FISH)   - Reading: Nature Education's Scitable: Clare O’Connor’s “Fluorescence In Situ Hybridization” Link: Nature Education's Scitable: Clare O’Connor’s “Fluorescence In Situ Hybridization” (HTML) 
 
Instructions: Please study the “In Situ Hybridization Is Used to Localize DNA Sequences on Chromosomes,” “Fluorescent Probes Are Introduced,” and “Using FISH to Identify the Positions of Genes” sections on this page.  Author Clare O’Connor works in the Department of Biology at Boston College.
 
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2.2 Nucleic Acid Amplification   2.2.1 Modifications of PCR   - Reading: Dr. Kary Banks Mullis’s “Polymerase Chain Reaction” Link: Dr. Kary Banks Mullis’s “Polymerase Chain Reaction” (HTML)
 
Instructions: Please read about Dr. Kary Bank Mullis.  It is rare that technological inventions receive a Nobel Prize, but Mullis was awarded the Nobel Prize in Chemistry in 1993 for his invention of the polymerase chain reaction (PCR) method.  Please study the main column and click on the “Questions About PCR” (PDF) link on the right and study that section as well.
 
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  • Reading: University of South Carolina's School of Medicine: Margaret Hunt’s “Real Time PCR” Link: University of South Carolina's School of Medicine: Margaret Hunt’s “Real Time PCR” (HTML)
     
    Instructions: Please study this page carefully.  You can learn about an advanced PCR application, which is essential in tissue specific gene expression applications.  Please note that the first step is to reverse translate RNA to DNA, because we can amplify only DNA in the test tube.  Because of the reverse transcriptase step, this method is also called RT-PCR.  Here, it gets little confusing since the method is in real time as well, because it follows the PCR product production in real time.  The best way to keep all of these aspects in mind is to remember this method as “real time RT-PCR.”  Please make sure that you click on the thumbprints to enlarge all the images embedded in the left maroon-colored panel of this site.  

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2.2.2 Subtractive Hybridization   - Reading: Evrogen’s “Suppression Subtractive Hybridization (SSH)” Link: Evrogen’s “Suppression Subtractive Hybridization (SSH)” (HTML)
 
Instructions: Please learn the content of this page.  Please note that subtractive hybridization is a technique that compares the differences between two nucleic acid pools and exponentially amplifies the differences in these pools by PCR.  The nucleic acid pools are commonly mRNA pools, thus such experiments generate subtraction cDNA libraries.
 
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  • Assessment: The Saylor Foundation: "BIO403 Unit 2.2 Assessments" Link: The Saylor Foundation: "BIO403 Unit 2.2 Assessments" (HTML)
     
    Instructions: You will find links to the following four assessments on this page: "PCR," "RT-PCR," "Subtractive Hybridization," and "Differential Screening."  These are multiple choice assessments with one correct answer.  Clicking on an answer will bring you to another page.  If your answer is correct, then it is acknowledged with a short explanation.  Please read the explanation carefully and proceed to the next assessment as prompted at the bottom of the page.  If you clicked on the wrong answer, then the click will bring you to a tutorial page.  Please study the tutorial page carefully.  At the end of the tutorial page, you will be prompted to return to the assessment and complete it again. You can also move between multiple choice pages using the links at the top of the page. Please complete the four assessments in order as the assessments are listed in increasing complexity.

2.3 Chemical Synthesis of Genes   - Reading: Wiley Online Library: Markus Fuhrmann, et al.’s “A Synthetic Gene Coding For the Green Fluorescent Protein (GFP) Is a Versatile Reporter in Chlamydomonas reinhardtii” Link: Wiley Online Library: Markus Fuhrmann, et al.’s “A Synthetic Gene Coding For the Green Fluorescent Protein (GFP) Is a Versatile Reporter in Chlamydomonas reinhardtii” (HTML or PDF)
 
Instructions:  You may read this as HTML or a PDF.  To access the PDF, go to “Article Tools” on the right side of the page and click on “Get PDF.”  This is a peer-reviewed publication.  Please study the last paragraph of the “Introduction,” the entire “Synthesis of a C. Reinhardtii-Adapted GFP” in the “Results” section, and the entire “Discussion” section on this page.  The aim of this project is to exchange the codons in the jellyfish GFP gene to codons that are more frequent in a unicellular alga.  Please note that the codon preference varies in different organisms. 
 
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  • Assessment: The Saylor Foundation: "BIO403 Unit 2.3 Assessments" Link: The Saylor Foundation: "BIO403 Unit 2.3 Assessments" (HTML)

    Instructions: You will find the following "Codon Preference" assessment here. This is a multiple choice assessment with one correct answer.  Clicking on an answer will bring you to another page.  If your answer is correct, then it is acknowledged with a short explanation.  Please read the explanation carefully.  If you clicked on a wrong answer, then the click will bring you to a tutorial page.  Please study the tutorial page carefully.  On the tutorial page, you will be prompted to return to the assessment and complete it again. You can also move back to the assessment main page using the link at the top of the page.

2.4 RNA Methods   2.4.1 Regulatory RNAs   - Reading: Nature Education’s Scitable: Suzanne Clancy’s “RNA Functions” Link:  Nature Education’s Scitable:  Suzanne Clancy’s “RNA Functions” (HTML)
 
Instructions: Please study this summary on the regulatory RNAs.  Here you will learn that besides the familiar mRNA, tRNA, and rRNA, other RNA functions are abundant in the cell.  The following sections will introduce you to several RNA techniques that are designed to modulate gene expression in the cell.
 
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2.4.2 RNAi (RNA Interference)   - Reading: Macalester College: Mary Montgomery’s “RNAi” Link: Macalester College: Mary Montgomery’s “RNAi” (HTML)
 
Instructions: Please study this page.  Please note that RNA interference is an experimental technique.
 
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2.4.3 Natural Ribozymes   - Reading: John W. Kimball’s Biology Pages: “Ribozymes” Link: John W. Kimball’s Biology Pages:  “Ribozymes” (HTML)
 
Instructions: Please study this page.
 
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2.4.4 Synthetic Ribozymes   - Reading: National Center for Biotechnology Information’s PubMed: American Society for Clinical Investigation: Nassim Usman and Lawrence M. Blatt’s “Nuclease-Resistant Synthetic Ribozymes: Developing a New Class of Therapeutics” Link: National Center for Biotechnology Information’s PubMed:American Society for Clinical Investigation:  Nassim Usman and Lawrence M. Blatt’s “Nuclease-Resistant Synthetic Ribozymes: Developing a New Class of Therapeutics” (HTML or PDF)
 
Instructions: Please study this publication on making synthesized ribozymes. This material is also available in PDF form from the top right corner of the page.
 
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2.4.5 Riboswitches   - Reading: National Center for Biotechnology Information’s PubMed: Journal ACS Chemical Biology: Shana Topp and Justin P. Gallivan’s “Emerging Applications of Riboswitches in Chemical Biology” Link:  National Center for Biotechnology Information’s PubMed: Journal ACS Chemical Biology:  Shana Topp and Justin P. Gallivan’s “Emerging Applications of Riboswitches in Chemical Biology” (HTML or PDF)
 
Instructions: Please study this page carefully.  This publication is challenging, but it helps you appreciate the regulatory roles of RNA in the cell.  Please rely heavily on the illustrations. It is not essential to memorize the names of the metabolites.  This material can also be viewed in PDF form from the top right corner of the page.  The authors work at Emory University.  This is a peer-reviewed publication.
 
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