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

Text Readings in Lewis

You may also ask questions and see answers to your classmates' questions in Web Crossing in the "Talk to Ross and Ed" discussion.

Answers to these questions and other helpful learning materials can be found on the web at the On-line Learning Center

Objectives

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.

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.

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.
• 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 Lewis' Text Chapter 13, pg 258-9, Biotechnology 13.2, (Recombinant DNA)
• Recombinant plasmids
• Incorporationg recombinant plasmids in bacteria
• *Recombinant DNA from DNA Interactive

  Choose "Techniques" at the bottom of the next 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)

• Examples of Restriction Enzymes

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 Center for Life Sciences, Colorado State University
• Transgenic rabbit Life, Lewis, et. al. Chapter 13 pg 256-7, Biotechnology 13.3, (Transgenes and Gene Therapy)
• Cloning and Transgenic Animals From Access Excellence
• The Land of Milk and Money From Scientific American
• More about the production and use of transgenic animals From Kimball's Biology Pages

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.

Kary Mullis - Dancing Naked in the Mind Field

• *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.

• Chapter 12, Biotechnology 12.3, PCR: An Application of Understanding DNA Replication, pgs 234-5
• Polymerase Chain Reaction Animation from:
• the Alternate Crops & Systems Lab of the United States Department of Agriculture
• Polmerase Chain Reeaction Animation from:
• Genetic Engineering Chapter of the MICROBIOLOGY 101/102 INTERNET TEXT by DR. RONALD E. HURLBERT from Washington State Unversity.
• PCR Diagram from The National Institutes of Healh

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.
• A PCR Thermal Cycler.
• 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 are being 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 Lewis, pg 238 for other examples.