Biology 100/101
Lecture 18
Biotechnology: Recombinant DNA, and PCR
(Print Version)

Announcements &
Assignments

Lecture Objectives

Recombinant DNA
Technology

Transgenic
Organisms

Polymerase
Chain Reaction
(PCR)

PCR has
Many Uses

Lecture Syllabus

IB 100/101 Home Page


Announcements

Text readings in Hoefnagels

Recombinant DNA Technology
Chapter 7, pg. 146-148
Chapter 14, pg. 252-257
Chapter 34, The Endocrine System, Diabetes Mellitus, pg 615

You have open access (no log-in or password needed) to instructional materials on the Text web site. Select "Resources" from the upper left of the page and select the text chapter you want.

Moodle

You may also ask questions and see answers to your classmates' questions in Moodle in the "Talk to Ed and Mike" forum.

Objectives

The content of today's lecture will help you answer question #2 on this assignment:

After studying this material you should be able to:

  1. Draw a diagram or write a description of the action of restriction enzymes in the cutting of DNA molecules into "restriction fragments".

  2. Outline a procedure for isolating a specific gene from a eukaryotic organism, incorporating it into a recombinant plasmid, and inserting it into a bacterial cell to produce a gene product.

    3. donor DNA restriction enzyme
    restriction site sticky ends
    plasmid recombinant DNA
    ligase vector
    Promoter

  3. Define and give an example of a transgenic organism.

  4. Describe how the polymerase chain reaction (PCR) is used to make millions of copies of specific sequences of DNA. Discuss the significance of PCR in the location of specific DNA sequences in a small sample of tissue.

    5. PCR
    polymerase chain Reaction    		 Thermus aquaticus
    and taq polymerase
    Yellowstone National Park high temperatures
    unzipping DNA DNA primers for specific DNA sequences
    DNA nucleotides binding or annealing of primers
    DNA replication repetition

Recombinant DNA Technology

  • Recombinant DNA Technology refers to molecular techniques that are used to insert DNA (genes) from one type of organism to another. For instance, the human gene for insulin production can be inserted into the DNA of a bacterial cell. The bacterial cell will then divide to produce many new bacterial cells, each with the gene for human insulin faithfully replicated. The bacteria then produces human insulin that can be harvested and used to treat people with diabetes.

  • Insulin example from Iowa Public Television

  • Bacteria have one large circular chromosome and several small circular pieces of DNA called plasmids.

  • Plasmids are naturally transferred from one bacterial cell to another. This is one way that bacteria can share genetic information in a primitive form of sex.

  • Antibiotic Resistance in Bacteria - Plasmids

  • Foreign genes can be artificially added to plasmids and placed into bacteria.

    • Restriction enzymes that cut DNA at very specific base sequences are used to cut open bacterial plasmids and the genes to be transferred leaving complementary "sticky ends" of DNA that allow the plasmid DNA and foreign DNA to be stuck together using an enzyme called ligase.

    • See Hoefnagels Text Chapter 12 - Transgenic Organisms, pg 252-254

    • *Recombinant DNA from DNA Interactive

      Choose "Techniques" at the bottom of the screen

      Choose "Cutting and pasting" at the top of the next screen

      Two animations, "Cutting and pasting DNA" and "Recombining DNA" are very useful.

  • Inserting a DNA Sample into a Plasmid (Access Excellence)

  • Plasmid Insertion (Access Excellence)

  • Transfer and Cloning of the Insulin Gene (Access Excellence)

DNA
(Human
Insulin Allele)
in Bacterial Plasmid		 -> Transcription
(in bacterium)		 -> Insulin
mRNA		 -> Translation
(in bacterium)		 -> Protein
(Insulin)
harvested
from bacteria

Transgenic Organisms, AKA Genetically Modified Organisms.

  • Transgenic is the word used to describe any organism which carries the genes (DNA) of another organism. Recombinant bacterial plasmids are one example, but the term is broad enough to include plants and animals that carry genes artificially inserted from another organism by any of a variety of methods.

  • Transgenic Crops From the Department of Soil & Crop Sciences, Colorado State University

  • Designing Genes for Insertion

    • Promoter - Transgene - Marker Gene - Termination Sequence

      Promoter - This is the on/off switch that activates the gene in the cells of the transgenic organism.

      Transgene - This is the gene that is being transferred from one organism to another.

      Selectible Marker Gene - This gene shows that the transgene construct is present and active in the cell of the tansgenic oraninsm. Marker genes may provide protection from an antibiotic or poison that allows researchers to kill cells that don't carry and express the transgene and retain those that do. Sometimes the marker gene causes a visible color change in the cells that express the transgene construct.

      Termination Sequence - This gene sequence marks the end of the gene where mRNA production (Transcription) should stop.

  • Transgenic glow-in-the-dark zebra fish (Hoefnagels, Chapter 12 pg 252, fig 12.19)

  • Transgenic Animals from Kimball's Biology Pages

  • EUROPEAN REGULATORS CURDLE PLANS FOR GOAT MILK HUMAN ANTITHROMBIN

Polymerase Chain Reaction (PCR)

  • The polymerase Chain Reaction is a biotechnology technique used to replicate or "amplify" a very specific portion of a much greater sample of DNA. The technique is similar to using a search engine like Google to find a specific document on the web and then print out many copies.

    • Try this exercise and see what happens. Use Google.com to do an advanced search of the web for this exact phrase, "You should learn the biological concepts that will help you make informed decisions".

    • What popped up? Google found that one document out of the millions of documents on the web using only 13 words.

  • Similar to the phrase you used in Google.com, single stranded DNA primers used in the polymerase chain reaction can find one specific complementary section of DNA or a specific gene among all the DNA of all 23 pairs of chromosomes and mitochondrial DNA in a DNA sample from a person. The technique uses laboratory constructed DNA primers that match specific, sequences of DNA that are known to precede and follow the section of DNA one wants to amplify.

    • When DNA is being replicated, people-made primers made of single stranded DNA provide the starting point for DNA polymerase (in this case Taq polymerase). An analogous situation is the requirement for transcription factors to provide the starting place for RNA polymerase in transcription.

    Once the primers locate the section of DNA the enzyme Taq polymerase, a DNA replication enzyme from Thermus aquaticus, a bacterium that lives in boiling hot springs in Yellowsone National Park, uses DNA nucleotides supplied by the researchers to produce billions of copies of that section of DNA in a matter of a few hours.
    chemistrydaily.com

    Kary Mullis - Dancing Naked in the Mind Field

  • Animation of the Polymerase Chain Reaction from sumanasinc.com.
    Select "The Polymerase Chain Reaction (PCR) ".
    Just watch the animation to get the "gist" of the concept of PCR.

  • *Polymerase Chain Reaction from DNA Interactive

      • Choose "Techniques" at the bottom of the next screen

      Choose "Amplifying" at the top of the next screen

      Two animations, "Making many copies of DNA" and "PCR animation" are very useful.

  • Polymerase Chain Reaction (Hoefnagels, pgs 146, fig 7.13)

PCR is used to amplify DNA for several purposes:

  • DNA from small samples of blood, tissue, or bodily fluids can be amplified for analysis in criminal cases.

  • Mitochondrial DNA from bone fragments and teeth can be used to identify relationships of the dead person to living realatives or ethnic groups.

  • Nuclear DNA from the victims of the September 11th attack on the World Trade Center were compared to DNA samples from living relatives or hair or other tissue samples supplied by families of the missing.

  • PCR primers matching DNA of specific species of organisms or viruses can be used to identify the presence of the organism or virus. Such tests are used to test for the presence of HIV in a blood sample or Anthrax spores on postal equipment.

  • See Hoefnagels, pg 146 for other examples.