DNA CLONING:THE BASICS AND THE

CONTROVERSYCreated by Alicia King

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 TC G A

G C TA 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

Molecular Cloning

Quiz!!!

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

ABC

CORRECT!

Great job! Click the arrow to move on to the next question!

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

ABCD

CORRECT!Great job!!

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

ABCD

CORRECT!Great job!!

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.

DOLLY THE SHEEP Watch this interesting news clip to learn more about Dolly!

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

Real world

Applications

Quiz!!!

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

CORRECT!Great job!

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

ABC

CORRECT!Great job!

INCORRECT Go back and try again!

QUESTION #3 What genes can be inserted into a

recombinant DNA sequence to make transected E. coli bioluminescence?

Lac ZDNA 1Lux

ABC

CORRECT!Great job!

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?

EXPANDING ON BIOETHICS Click anywhere in the red box to begin this short video about

bioethics!

ETHICS QUIZ!

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

AB

C

D

CORRECT!Great job!

INCORRECT Go back and try again!

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

AB

C

D

CORRECT!Great job!

INCORRECT Go back and try again!

QUESTION #3 Bioethics is:

Not controversialA rapidly emerging fieldThe study of ethics brought about

by advancements in scienceB and CAll of the above

ABC

DE

CORRECT!Great

job!

INCORRECT Go back and try again!

QUESTION #4 Is reproductive cloning ethical?

TRUE FALSE

CORRECT! There is actually no right or

wrong answer here, as long as you can support your opinion!

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!!!

I, , successfully completed the lesson module DNA Cloning: The basics and the controversy on . I know the difference between molecular and reproductive cloning, as well as know some of the bioethical arguments for both sides of the debate.

X

Congratulations!