We are working with our class textbooks and on our computers to learn about the techniques used in biotechnology. It is REALLY important that you read the information in your textbook before viewing the animations. Refer to the "Objective" statements in each section to make sure you have understood each technique fully.
The handout of summary questions that you received in class is due on Friday.
Self-directed tutorial on DNA Technology
The handout of summary questions that you received in class is due on Friday.
Self-directed tutorial on DNA Technology
DNA technology is an evolving field. There are lots of resources available that will help you more than just reading in a book. I’ve put together this tutorial to give you the opportunity to see the procedures in action through animations and, in some cases, to perform simulations. These exercises are not meant to stand alone. Use your book to support your understanding of the topics described in this tutorial as you proceed. You may go at your own pace as long as you complete all activities by the due date. There are several activities that we will be completing as a whole class.
Topic 1: DNA Cloning (this topic is covered on pp. 385 – 392 of your text):
Background: DNA cloning permits production of multiple copies of a specific gene or other DNA segment. This can be accomplished using a bacterial plasmid, polymerase chain reaction, and a few other techniques we will leave for another course. Why do scientists need to clone DNA? Here are a few of the many reasons: to produce a protein product (ex. human growth hormone), to provide an organism with a new metabolic capability (ex. pest resistance), or to produce enough copies of the DNA for analysis (ex. at crime scenes).
THE TECHNIQUES
Restriction Enzymes (p. 386) – these are needed to make DNA that contains nucleotide sequences from two different sources. For example, if we want to insert a gene for human growth hormone into a bacterial plasmid we would use restriction enzymes. The bacteria will then produce the human growth hormone, a protein, as part of its regular metabolism.
Objectives
After completing the restriction enzyme section (animation AND reading in the text) the student should be able to:
- Describe the natural source and function of restriction enzymes.
- The student should be able to understand why restriction sites need to be palindromic.
- The student should be able to explain how restriction enzymes can be used to combine DNA from two different sources to make recombinant DNA.
1. Online go to http://www.dnai.org/b/index.html (you might want to bookmark this – we will be going back to it several times)
At this website, click on “techniques” (the bottom middle button).
From there, click “cutting & pasting” at the top of the slide.
a. View the animation for “Cutting and pasting DNA”
b. Then watch the animation of “Recombining DNA”.
READ THE ACCOMPANYING TEXT to understand what is going on and why!!!
Bacterial plasmids (pp. 386-388) – these are small circles of extrachromosomal DNA found in many types of bacteria. Plasmids are often used because they can be easily isolated from bacteria, manipulated to form recombinant plasmids by insertion of foreign DNA, and then reintroduced into bacterial cells. Moreover, bacterial cells reproduce rapidly, multiplying any foreign DNA they carry.
Objectives
After completing the restriction enzyme section (animation AND reading in the text) the student should be able to:
- Explain how vectors are used in recombinant DNA technology
- Suggest limitations of using bacterial vectors to create mammalian proteins
1. Online view the animation about cloning genes using bacterial plasmids at http://www.sumanasinc.com/webcontent/animations/content/plasmidcloning.html
2. (practice it) Read and complete the Worksheet entitled “Gene Cloning”
3. Online, learn about cloning genes by going to http://www.dnai.org/b/index.html
Once again, click on “techniques.”
From there, click “Transferring and Storing.”
Watch the animation for “Transformation.”
Forming cDNA (p. 390) – one of the problems with using bacteria to produce human proteins is that bacteria do not have introns within their genes. They do not have a mechanism to get rid of the introns that are within the human gene. To get around this, scientists produce complementary DNA (cDNA). This process uses a messenger RNA transcript from the gene (so the introns have been removed) and the enzyme reverse transcriptase to produce DNA. This can then be inserted into the plasmid.
Objectives
After completing the restriction enzyme section (animation AND reading in the text) the student should be able to:
- Give a step-by-step description of how cDNA is created.
- Explain why we need to use cDNA when bacteria are the vector
- Explain the role of reverse transcriptase in creating recombinant DNA
Creating a DNA Library (pp. 388-390) – a DNA library, or genomic library, is the complete set of plasmid clones, each carrying copies of a particular segment from the initial genome.
Objectives
After completing the restriction enzyme section (animation AND reading in the text) the student should be able to:
- Define and distinguish between genomic libraries using plasmids, phages, and cDNA.
1. Here is how it is done (click on either “step through” or “narrated”): http://www.sumanasinc.com/webcontent/anisamples/nonmajorsbiology/dnalibrary.html
Once again, click on “techniques.”
From there, click “Transferring and Storing.”
Watch the animation for “Storing”
PCR (pp. 391-392) – Polymerase Chain Reaction allows for very quick amplification (many copies) of any specific DNA target segment. The process takes place in a test tube, so no living organisms are required. With automation, PCR can make billions of copies of a target segment of DNA within a few hours.
Objectives
After completing the restriction enzyme section (animation AND reading in the text) the student should be able to:
· Describe the polymerase chain reaction (PCR) and explain the advantages and limitations of this procedure.
2. Then try this other simulation at http://www.dnai.org/b/index.html
Click on “techniques” then “amplifying.”
Perform the interactive tutorial entitled “Making Many Copies of DNA”
Topic 2: Restriction Fragment Analysis (pp. 392-394) –
Background: This can be used to detect DNA differences that affect restriction sites. Some applications of restriction fragment analysis are: to look for variations in genes from person to person, to identify whether a particular allelic change is associated with a hereditary disorder, to find where a gene is located in the genome, and to compare the same gene from species to species to investigate evolutionary relationships.
THE TECHNIQUES
Gel Electrophoresis –this process is used to separate molecules based on size and charge.
Objectives
After completing the restriction enzyme section (animation AND reading in the text) the student should be able to:
· Explain how gel electrophoresis is used to analyze nucleic acids and to distinguish between two alleles of a gene.
1. Online, review gel electrophoresis by going to http://www.dnai.org/b/index.html
Click on “Techniques” and then the “sorting and sequencing” button.
Watch the animation titled “gel electrophoresis.”
2. (Practice It) Complete the activity “DNA Goes to the Races”
3. (Practice It) Complete the questions from the worksheet entitled “Restriction Maps and Logic Puzzles”
Southern Blot – this technique uses a combination of gel electrophoresis and then a specific probe to identify sequences within the DNA.
Objectives
After completing the restriction enzyme section (animation AND reading in the text) the student should be able to:
- Describe the Southern blotting procedure and explain how it can be used to detect and analyze instances of restriction fragment length polymorphism (RFLP)
1. Watch and read this animation that follows both gel electrophoresis and southern blotting: http://bcs.whfreeman.com/thelifewire/content/chp16/1602001.html
2. Try your hand at the following online quiz after watching the animation http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_5.html
Topic 3: Genomics – this is the study of whole sets of genes and their interactions.
THE TECHNIQUES
DNA Microarray (pp. 400 – 401) – consists of tiny amounts of a large number of single stranded DNA fragments representing many genes fixed to a glass slide in a tightly spaced array (grid). This is used to measure the expression of thousands of genes at one time.
Objectives
After completing the restriction enzyme section (animation AND reading in the text) the student should be able to:
- Explain the purposes of gene expression studies. Describe the use of DNA microarray assays and explain how they facilitate such studies.
1. Online, go to http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#
Select “Microarray” from the list to see how a DNA microarray is created (although all of the other animations are very good, too).
2. Watch the interactive animation of the process at http://www.bio.davidson.edu/courses/genomics/chip/chipQ.html
3. Great interactive! http://learn.genetics.utah.edu/units/biotech/microarray/
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