NATVIGATION Before we begin, it is important to
understand how to travel around this lesson!
Just click on these buttons throughout the Powerpoint to direct yourself to the appropriate places!
Advance to the next slide
Go back to the previous slide
Return to the Main MenuClick on me!
HELLO STUDENTS! This lesson is intended for grades 9-10 In order to access all of the activities
and information, you need to be hooked up to internet access
OBJECTIVES There are three main objectives behind
this lesson. After completion, you will be able to:Organize the basic steps for molecular
cloning. Identify the three basic steps used for
reproductive cloning.Differentiate between pros and cons of
reproductive cloning.
WHY DOES IT MATTER? Have you ever taken
an antibiotic?
Do you enjoy watching TV shows about crime scene investigation?
Some type of cloning technology is involved in these situations!
You should already be familiar with:Basic DNA structureThe function of nucleotidesDNA is the basis for life and unique genetic
material
Answer just a couple of review questions to check your understanding on those topics.
IN ORDER TO CONTINUE…
CHECKING UNDERSTANDING OF PREVIOUS MATERIAL #1
Click on the letter matched with the nucleotide bases as you would find in a piece of double stranded DNA.
(different shades of blue correspond to different strands)
G C T
C G A
G C T
A T C
A
B
GREAT JOB!You accurately recalled which nucleotide
bases paired together on complementary strands of DNA as found within our genome.
Not quite
Recall how bases pair:Adenine (A) pairs with Thymine (T), while
Cytosine (C) pairs with Guanine (G)
Using that information, go back and try again.
CHECKING UNDERSTANDING OF PREVIOUS MATERIAL #2 Below is the basic structure of a nucleotide of DNA.
There are three main parts: a phosphate group, a pentose sugar, and a nucleotide base.
Click on the arrow corresponding to the part that is responsible for creating unique DNA sequences across organisms.
Below are the correctly labeled parts of the nucleotide.
GREAT JOB!
Nucleotide base
Pentose sugar
Phosphate group
Not quite
The phosphate and pentose sugars create the back bone of DNA structure and are not unique across individuals.
Using that information, go back and try again.
WHAT IS COMING UP… Great job on completing the review
questions successfully! The next slide includes a main menu for
the lesson. There are three main parts. You should complete them in this order:
1. The Basics of Molecular Cloning2. Real World Applications3. Bioethics
On Main Menu, just click on the box corresponding to the first section to begin!
MAIN MENUDNA
Cloning:The Basics
and the Controvers
y
1. The Basics
of Molecular Cloning
2. Real World
Applications
3. Bioethics
OVERVIEW In this section, you will first view a
series of slide containing information about molecular cloning.
Then, you will answer a few questions to check for your understanding on this section before moving onto the Real World Applications section.
WHAT IS MOLECULAR CLONING? Molecular cloning is the process by
which one molecule is multiplied INTO other molecules
This means that the main goal is to insert a DNA fragment of interest into a plasmid vector. A plasmid vector is a piece of circular DNA
that can replicate on its own. After the plasmid vector takes up the
new fragment, it can be amplified in host bacterial cells.
GENERAL APPLICATIONS Common uses for this type of cloning
includes:Protein productionGenetic fingerprintingSequencing of entire genomesDiagnostic testing to identify
an illness based on a recombinant protein
Many other biological experiments!Genetic fingerprinting involves separating sequences of DNA based on size.
STEP-BY-STEP The following slides outline these basic
steps in the molecular cloning process1. Fragmentation2. Ligation3. Transfection4. Screening/Selection
FRAGMENTATION This first step is the
process by which a strand of DNA is broken apart into pieces, or fragments.
A plasmid is also cleaved at the same recognition sites on the DNA.
HOW DOES THE DNA GET BROKEN APART? These fragments are isolated by the use of
a restriction endonuclease enzyme. These enzymes cleave (cut) DNA molecules only where specific DNA sequences are located.
Complementary “sticky ends: are created on both the DNA fragment to be cloned and the plasmid vector.
Original double stranded DNA
LIGATION In this second step, fragments are placed, or
‘glued’, together in the desired sequence at complementary sticky ends.
Ligase is an enzyme that accomplishes this by catalyzing the formation of chemical bonds between two pieces of DNA.
The end result of this step results in recombinant DNA.
Ligase
(plasmid)
Isolated DNA fragment to be cloned
TRANSFECTION In this third step, the
newly created recombinant DNA is inserted into host cells.
Qualities of a good host include: Cheap and easy to
reproduce Easily uptake the
recombinant DNA Support the growth of
the recombinant DNA
One of the most commonly used host cell used is the Escherichia coli, known as E.coi, is shown here.
SCREENING/ SELECTION In this final step, cells that have been
successfully transfected into hosts are selected.
One of the most common ways to select for the cloned cells of interest is to grow them on agar (the gel like material in petri dishes that allows for bacterial growth) with selective nutrients.
MOLECULAR CLONING IN E. COLI Recombinant DNA with the lux genes
taken up by E. coli can make the E. coli cells bioluminescence (a.k.a. glow in the dark)!
Bioluminescent E. coli would look something like this under a black light.
HYPOTHETICAL MOLECULAR CLONING APPLICATION Some bacteria is capable of growing only on glucose.
Which of the following petri dishes might you expect to see if you had successful transduction and selection of a gene that inhibited bacteria from growing with glucose, but enabled it to grow with lactose instead. You are trying to select for the cloned bacteria. Successfully cloned (recombinant) bacteria = red Unsuccessfully cloned bacteria = yellow
Click on the correct plate.
Plate results:
Agar nutrients: Lac only Lac only Glu only
YOU GOT IT! The Lac only plate you chose with only
the red colonies was correct because:The original (yellow) bacteria cannot grow
on lactoseThe recombinant (red) bacteria can now
only grow on lactose, not on glucose anymore
Great job!
HMMM…. Let’s think about this one again…
Remember that the original (red) bacteria can only grow on glucose
Also remember that the recombinant (yellow) bacteria can only grow on lactose now
REVIEW Now, it’s time to test your knowledge on
molecular cloning before moving on! Answer the following three practice
questions until you get them 100% correct!
To get feedback on the questions, simply click on your answer.
QUESTION #1 Correctly order the steps in molecular
DNA cloning:1-fragmentation2-tranfection3-screening4-ligation
A) 1, 2, 4, 3B) 1, 4, 3, 2
1, 4, 2, 3
A
B
C
INCORRECT Go back and try again!
Remember that the final step is to isolate the cells with successful uptake of the cloned DNA.
QUESTION #2 The goal of the ligation step in
molecular cloning is to:create fragmentsglue DNA fragments togetherselect for cells that have the new DNAinsert recombinant DNA into host
cells
A
B
C
D
INCORRECT Go back and try again!
Remember that ligation is the second step in the molecular cloning process.
QUESTION #3 The purpose of the restriction
endonuclease is to:A) glue fragments back togetherB) select for cloned cellsC) cleave DNA at specific sequences
none of the above
A
B
C
D
INCORRECT Go back and try again!
Remember that restriction endonucleases are used during the fragmentation step.
CONGRATULATIONS! You have successfully completed this
first section on Molecular Cloning! Go back to the Main Menu and select
the next section, Real World Applications.
OVERVIEW First, you will look at an example of
molecular cloning in medicine. Next, you will learn about the basics of
reproductive cloning. Then, you will look at Dolly the Sheep as
an example of reproductive cloning. Finally, you will complete a short review
about these real world applications!
REAL WORLD MOLECULAR CLONING One way molecular cloning can benefit
medicine is by producing antibodies to help fight disease.
A video on the next slide goes over the basic steps of how this happens.
MOLECULAR CLONING VIDEO CLIP Click on the play button to watch this genetic engineering
example of molecular cloning.
REPRODUCTIVE CLONING This is they type of cloning most commonly
thought about as it is the type of cloning that creates an animal with the same nuclear DNA as another animal.
Nuclear DNA is contained within the nucleus of eukaryotic organisms and encodes the genome.
Dolly the Sheep is one of the most successful stories of reproductive cloning.
BASIC STEPS IN REPRODUCTIVE CLONING1. Isolate donor nucleus
of an adult animal (to be cloned).
2. Isolate an egg from another adult animal by removing the nucleus.
3. Fertilize that egg with the isolated donor nucleus.
4. The fertilized cell is placed in a host uterus until birth.
(Note that even though this is shown as a sheep example, the same steps can be applied to other animals.)
1
2
4
3
1
2
3
4
REVIEW It’s time to test your knowledge again!
This time, the questions over the section you just completed on real world applications and reproductive cloning.
Answer the following three practice questions until you get them 100% correct!
QUESTION #1 True or False: Reproductive cloning will
never be able to produce new organs to possibly treat or cure diseases.
TRUE FALSE
INCORRECT Go back and try again!
Remember that technology is improving every day, just as it did before the reproduction of Dolly!
QUESTION #2 Order these basic steps of reproductive
cloning (currently in no particular order).1-fertilized cell placed in host uterus2-isolate DNA to be cloned from an adult nucleus3-place DNA to be cloned into a cell lacking a nucleus which will be fertilized
A) 3, 1, 22, 3, 12, 1, 3
A
B
C
QUESTION #3 What genes can be inserted into a
recombinant DNA sequence to make transected E. coli bioluminescence?
Lac ZDNA 1Lux
A
B
C
INCORRECT Go back and try again!
Recall that bioluminescence was the example of molecular cloning we discussed.
CONGRATULATIONS! You have successfully completed this
first section on Real World Applications! Go back to the Main Menu and select
the next section, Bioethics.
OVERVIEW In this section, you will learn about
some of the ethical arguments behind cloning.
Once you have completed the informational section, you will again complete a few review questions.
As you are learning about the ethics of cloning, be considering everything in order to form your own opinions
WHY DOES IT MATTER? Reasons cloning is a debatable issue:
Religious or moral grounds against cloningMany countries have even banned cloning
on these groundsEnvironment has a lot to do with the make
up of person/animal, so there are other confounding factors into creating a clone beyond genetics
Some view cloning as a type of selective breeding
PRO ARGUMENTS Could help cure diseases through the
genetic engineering of cells, tissues, and organs through therapeutic cloning
Could help better understand and combat genetic diseases
Could be another solution for infertile couples
MORE PRO ARGUMENTS… Could potentially select for specific
traits in organisms that could be beneficial in research
Cloning plants and animals could potentially provide an indefinite, healthy food source
The realms of science should not be restricted
CON ARGUMENTS Would decrease genetic diversity,
thus also weakening ability for adaption
Safety is a concern Could be a malpractice disaster as
there could be unforeseen health risks or outcomes
MORE CON ARGUMENTS… Someone does not have the right to
genetically engineer another being Creating and destroying embryos
through this process is unethical (typically a religious argument)
Many legal arguments, such as would a cloned being have the same rights as those traditionally reproduced?
REVIEW It’s time to test your knowledge again!
This time, the questions over the section you just completed on bioethics.
Answer the following three practice questions until you get them 100% correct!
Note: the answers to these questions are meant to be unbiased and should reflect what was presented in this lesson.
QUESTION #1 What is a pro(s) in the ethical cloning
debate?
potential to regenerate organscould be a solution for infertile
couplesscience should not be restricted all of the above
A
B
C
D
QUESTION #2 What is a con(s) in the ethical cloning
debate?
Increased threat of malpracticecould decrease genetic
diversitydestroying embryos is
unethicalall of the above
A
B
C
D
QUESTION #3 Bioethics is:
Not controversialA rapidly emerging fieldThe study of ethics brought about
by advancements in scienceB and CAll of the above
A
B
C
D
E
You have successfully completed this introductory lesson about DNA cloning!
On the next slide, type in your name and date. Then print and sign your name to
assert your completion!
WAY TO GO!!!
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