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Dna cloning: The basics and the controversy

Dna cloning: The basics and the controversy. Created 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

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Dna cloning: The basics and the controversy

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  1. Dna cloning:The basics and the controversy Created by Alicia King

  2. 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 Menu Click on me!

  3. 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

  4. 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.

  5. 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!

  6. In order to continue… • You should already be familiar with: • Basic DNA structure • The function of nucleotides • DNA is the basis for life and unique genetic material • Answer just a couple of review questions to check your understanding on those topics.

  7. 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) A B

  8. Great job! You accurately recalled which nucleotide bases paired together on complementary strands of DNA as found within our genome.

  9. 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.

  10. 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.

  11. Great job! • Below are the correctly labeled parts of the nucleotide. Nucleotide base Phosphate group Pentose sugar

  12. 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.

  13. 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 Cloning 2. Real World Applications 3. Bioethics • On Main Menu, just click on the box corresponding to the first section to begin!

  14. Main Menu 1. The Basics of Molecular Cloning DNA Cloning: The Basics and the Controversy 3. Bioethics 2. Real World Applications

  15. The Basics of Molecular Cloning

  16. 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.

  17. 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.

  18. General applications • Common uses for this type of cloning includes: • Protein production • Genetic fingerprinting • Sequencing of entire genomes • Diagnostic testing to identify an illness based on a recombinant protein • Many other biological experiments! Genetic fingerprinting involves separating sequences of DNA based on size.

  19. Step-by-step • The following slides outline these basic steps in the molecular cloning process 1. Fragmentation 2. Ligation 3. Transfection 4. Screening/Selection

  20. 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.

  21. How does the DNA get broken apart? • These fragments are isolated by the use of a restriction endonucleaseenzyme. These enzymes cleave (cut) DNA molecules only where specific DNA sequences are located. Original double stranded DNA Complementary “sticky ends: are created on both the DNA fragment to be cloned and the plasmid vector.

  22. 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. Isolated DNA fragment to be cloned (plasmid) Ligase

  23. 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.

  24. 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.

  25. 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.

  26. 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

  27. You got it! • The Lac only plate you chose with only the red colonies was correct because: • The original (yellow) bacteria cannot grow on lactose • The recombinant (red) bacteria can now only grow on lactose, not on glucose anymore • Great job!

  28. 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

  29. Molecular Cloning Quiz!!!

  30. 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.

  31. Question #1 • Correctly order the steps in molecular DNA cloning: 1-fragmentation 2-tranfection 3-screening 4-ligation A) 1, 2, 4, 3 B) 1, 4, 3, 2 1, 4, 2, 3 A B C

  32. CORRECT! Great job! Click the arrow to move on to the next question!

  33. Incorrect Go back and try again! Remember that the final step is to isolate the cells with successful uptake of the cloned DNA.

  34. Question #2 • The goal of the ligation step in molecular cloning is to: create fragments glue DNA fragments together select for cells that have the new DNA insert recombinant DNA into host cells A B C D

  35. CORRECT! Great job!!

  36. Incorrect Go back and try again! Remember that ligation is the second step in the molecular cloning process.

  37. Question #3 • The purpose of the restriction endonuclease is to: A) glue fragments back together B) select for cloned cells C) cleave DNA at specific sequences none of the above A B C D

  38. CORRECT! Great job!!

  39. Incorrect Go back and try again! Remember that restriction endonucleases are used during the fragmentation step.

  40. 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.

  41. Real World Applications

  42. 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!

  43. 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.

  44. Molecular cloning video clip • Click on the play button to watch this genetic engineering example of molecular cloning.

  45. 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.

  46. Dolly the sheep • Watch this interesting news clip to learn more about Dolly!

  47. Basic Steps in reproductive cloning 1 • Isolate donor nucleus of an adult animal (to be cloned). • Isolate an egg from another adult animal by removing the nucleus. • Fertilize that egg with the isolated donor nucleus. • 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 3 2 3 4 4

  48. Real world Applications Quiz!!!

  49. 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!

  50. Question #1 • True or False: Reproductive cloning will never be able to produce new organs to possibly treat or cure diseases. TRUE FALSE

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