Genetic Engineering and Recombinant DNA

1. Genetic Engineering and Recombinant DNA

2. Genetic Engineering and Recombinant DNA • The Origin of Genetic Engineering • Biotechnology - the use of living organisms for practical purposes. • While many believe that biotechnology is a novel concept, it actually began about 10,000 years ago when human populations began selecting and breeding useful plants, animals, fungi, and microorganisms. While the early biotechnology techniques were relatively simple, modern genetic engineers move genes among all kinds of organisms, including humans, mice, tomatoes, yeasts, and bacteria.

3. Knowing Biochemical Pathways Helps Molecular Biologists Design Useful Organisms • Today, the knowledge of biochemical pathways in some organisms allows biologists to predict what type of mutation will produce a desired trait. • With this in mind, molecular biologists have been successful in designing useful organisms by inserting or destroying genes that code for proteins involved in specific biochemical pathways. What are some examples of this technology?

4. Knowing Biochemical Pathways Helps Molecular Biologists Design Useful Organisms • For example, Calgene in central California deliberately damaged the gene that controls ethylene production in tomatoes. • Eythlene is responsible for fruit ripening. • Since these tomatoes do not produce ethylene, they will only ripen after the tomato distributor sprays them with ethylene. • This prevents the tomatoes from being picked before they have developed their flavor components. Such tomatoes are termed “Flavor-Saver” tomatoes.

5. How Do Restriction Enzymes Cut Up a Genome? • DNA can be “cut” with special enzymes termed endonucleases. • Endonucleases recognize specific sequences of nucleotides and sever the DNA at these sites. • Endonucleases evolved in bacterial cells as a defense against bacteriophages (bacterial viruses). • When phage DNA enters a bacteria, endonucleases break down the phage DNA (by cutting) in order to restrict viral replication. • Since endonucleases restrict viral replication, they have become known as Restriction Enzymes.

6. How Do Restriction Enzymes Cut Up a DNA? • Restriction enzymes recognize and “cut” DNA that is foreign to the bacterial cell. • The DNA of the bacterial cell is chemically modified to prevent attack by restriction enzymes. • Restriction Enzymes, therefore, “chop-up” foreign DNA, while leaving the DNA of the bacterial cell unaffected.

7. How Do Restriction Enzymes Recognize sites for severing? • Since their discovery in 1962, hundreds of restriction enzymes have been identified and isolated from bacterial cells. • These restriction enzymes are extremely specific and work by recognizing short nucleotide sequences in DNA molecules termed RECOGNITION SEQUENCES. • Once these sequences are detected, the restriction enzyme severs the DNA at this point.

8. EXAMPLE • Hae III cuts at the following recognition sequence: • GGCC • CCGG • Hae III will cut the DNA every time the above recognition sequence is detected. • The result is a matching set of restriction fragments. • Restriction fragments are pieces of DNA that begin and end with a restriction site.

9. Hae What? Hae III cuts the DNA each time the recognition sequence repeats itself within a DNA sample.

10. Mapping • A comparison of restriction fragment sizes allows biologists to construct a restriction map. • Restriction maps demonstrate how the restriction sites are placed within a piece of DNA. • More importantly, biologists can join these fragments into new combinations. • For example, human and mouse fragments can be joined together.

11. DNA Fingerprinting Example: Suppose Joe’s DNA has four restriction sites for EcoR1; EcoR1 will, therefore, cut Joe’s DNA four times _______________________________________________ 5 fragments result from the action of EcoR1when applied to Joe’s DNA What is the restriction site for EcoR1?

12. DNA Fingerprinting Example: Suppose Anisa’s DNA has 3 restriction sites for EcoR1. EcoR1 will, therefore, cut Anisa’s DNA three times. _______________________________________________ 4 DNA segments result

13. Restriction Enzyme & DNA Fragments EcoR1 cuts Joe’s DNA into 5 fragments and Anisa’s into 4. Joe Anisa Note: In addition to differing in fragment number, the size of the fragments differs as well. Why is this significant?

14. These fragments can now be separated from one another using ELECTROPHORESIS • DNA electrophoresis utilizes an agarose gel and a voltage current to separate the cut DNA fragments from one another. • The DNA samples are placed into the agarose gel (a medium in which the DNA fragments will travel) and the voltage current separates the fragments. • How?

15. Gel Electrophoresis - + Joe’s DNA Anisa’s DNA The current is applied and the fragments travel to the + end due to the negatively charged DNA (phosphate).

16. - + Joe’s DNA Anisa’s DNA Analysis Smaller DNA fragments will travel farther on the gel than larger DNA fragments.

17. Fingerprinting • Since every individual has a unique sequence of bases in their DNA, a unique banding pattern will be generated by electrophoresis for each individual. • This is known as a GENETIC FINGERPRINT. • NOTE: Even if two individuals have the same number of restriction sites in their DNA, the size of each fragment will differ and will, therefore, yield a unique banding pattern. • The next slide presents an example:

18. DNA from hair found on victim DNA from blood sample suspect #1 DNA from blood sample suspect #2 DNA from blood sample suspect #3 Forensics Who did it? Where is the heaviest band? Where is the lightest band? All 4 samples are cut with the same restriction enzyme. How many restriction sites does the DNA from suspect #1 have?

19. How Do Molecular Biologists Use Recombinant DNA? • Recombinant DNA - a DNA molecule consisting of two or more DNA segments that are not found together in nature. • For example, the next slide demonstrates how cells from a tobacco plant are infected with a plasmid carrying a gene for herbicide resistance. The herbicide resistant cells grow into mature plants which produce seeds containing the resistant gene.

20. Genetic Engineering and Recombinant DNA How Do Molecular Biologists Use Recombinant DNA?

21. How Do Molecular Biologists Use Recombinant DNA? • Recombinant DNA has provided scientists with: • 1) a tool for studying structure, regulation and function of individual genes; • 2)a tool for unraveling the molecular bases of molecular diseases; • 3)the ability to turn organisms into factories that turn out vast quantities of product (protein or other substance) that these organisms would never make on their own.

22. How Do Molecular Biologists Join Restriction Fragments Together? • Two pieces of DNA from different sources can be linked together by the enzyme DNA ligase. • DNA ligase is normally used during DNA replication. • DNA ligase is responsible for the linkage of separate pieces of DNA into one continuous strand.

23. Ligase This image demonstrates how ligase can be used to link human and mouse DNA together as well as the insertion of the human insulin gene into a plasmid causing the bacteria to produce insulin.

24. How Do Molecular Biologists Express Recombinant DNA in Bacteria and Other Hosts? • Molecular biologists face two serious challenges: • 1) To produce large numbers of particular genes. • 2) To induce host cells to express recombinant genes as usable proteins.

25. How Can Bacteria Be Induced To Make Great Numbers of Copies of a Gene? • Biologists achieve this goal with the use of plasmids. • Plasmids allow bacterial cells to produce large numbers of copies of a single gene. • Using DNA ligase, researchers can link any gene to a plasmid which carries recombinant DNA into cells. • Plasmids are an example of a vector. • A vector is anything that spreads genes from one organism to another.

26. How Can Bacteria Be Induced To Make Eukaryotic Genes? • Eukaryotic DNA contains introns which are base sequences in the pre-mRNA that are not expressed and normally removed by the eukaryotic cell before the mRNA is translated. • Bacterial cells are prokaryotic and, therefore, do not have the required enzymes to recognize and remove the introns. • If they cannot remove introns, they cannot make a mRNA molecule that is translateable and, therefore, cannot directly make eukaryotic genes.

27. How Can Bacteria Be Induced To Make Eukaryotic Genes? • The solution of intron removal in bacterial cells comes from the action of retroviruses. • Recall that retroviruses contain reverse transcriptase which allows the conversion of RNA to DNA. • Researchers can take mature mRNA (introns have already been removed) and copy it back to DNA with the use of reverse transcriptase. • The resulting DNA is termed complementary DNA (cDNA) and, unlike the genomic DNA, it has no introns.

28. The image to the left demonstrates how reverse transcriptase is used to copy mature insulin mRNA into DNA. This DNA can now be joined to a plasmid vector and expressed by a bacterium.

29. Can Host Cells Be Induced To Express Polypeptides in a Usable Form? • Unfortunately, not all eukaryotic genes can be expressed in bacteria. • Such genes code for proteins that must be modified after translation. • For example, most membrane proteins require modifications that can only be made in eukaryotic hosts.

30. How Do Researchers Make Multiple Copies of Recombinant DNA? • Researchers need enormous quantities of a gene in order to sequence it, detect mutations or study how proteins interact with the gene to influence gene expression. • Cloning and PCR(Polymerase Chain Reaction) allow researchers to make millions of copies of a particular gene.

31. How Do Researchers Make Multiple Copies of Recombinant DNA? Cloning simply involves the introduction of a single recombinantDNA (gene and plasmid) molecule into a bacterial host cell. The plasmid can induce the host cell to make many copies of the gene it carries and, in addition, researchers can induce the bacterial cell to divide rapidly. As the bacteria divide, the recombinant DNA multiplies.

32. PCR allows researchers to produce multiple numbers of individual DNA sequences in a very short period of time. • In PCR: • The selected DNA segment is heated causing the two strands to separate. • The DNA is cooled and two short nucleotide sequences termed primers bind to the complementary DNA strands. • DNA polymerase then copies each strand until the researcher stops the reaction by again raising the temperature. • Increasing the temperature repeats the process.

33. How Do Biologists Find the Right DNA Sequence in a Recombinant DNA Library? • A gene library is a collection of restriction fragments from a single genome. • Such a library is only useful to researchers if they can find the gene they are interested in.

34. Two tools are used to find specific genes: • 1) hybridization probes - short segments of single stranded DNA that binds to and detects the gene in question. • 2) antibodies - detect and bind with specific proteins in colonies of bacteria containing recombinant DNA. The following slides demonstrates the use of each technique.

35. Genetic Engineering and Recombinant DNA Note that the hybridization probe locates specific DNA sequences while antibodies locate the protein product of the same sequence. Figure 13-4

36. Genetically Engineered Bacteria and Eukaryotic Cells Can Make Useful Proteins • Genetic reprogramming using recombinant DNA technology allows the production of an extraordinary number of products. • For example: • insulin • growth hormone • ingredients for processed foods • enzymes used to produce valuable molecules or destroy pollutants • enzymes in laundry soap • Vaccines • New proteins researchers are currently developing new antibodies that can interfere with disease processes

37. Gene Therapy: • Products of Recombinant DNA Can Be Released Directly into the Body from Engineered Somatic Cells • Gene Therapy - The insertion of therapeutic genes into an individual so that their products act to modulate a particular phenotype. • One strategy associated with gene therapy involves the removal of cells from the body, engineering them to produce the desired effect, and then implanting them back into the body of the individual. • For example, researchers are now experimenting with the insertion of genes for clotting factor into cells that are then implanted into individuals suffering from hemophilia. • This allows the body to produce clotting factor and alleviate symptoms associated with hemophilia.

38. RECOMBINANT DNA CAN GENETICALLY ALTER ANIMALS AND PLANTS • Organisms that carry recombinant DNA are termed transgenic organisms and the added DNA is termed a transgene.

39. How Do Researchers Produce a Transgenic Mammal? • For a gene to be expressed, researchers must put the transgene into the zygote before the beginning of embryonic development. • If this is performed successfully, all of the cells of the organism will contain the desired DNA. • To date, researchers have been successful in producing transgenic mice, pigs, goats, and sheep.

40. How Do Researchers Produce a Transgenic Mammal? • The engineering of transgenic animals faces serious obstacles: • 1) they must be made one at a time; • 2) in knockouts (animals in which a particular gene has been inactivated), recombinant genes are inserted at random and may not function as researchers hope. • In spite of these obstacles, such animals can provide clues about how previously mysterious proteins function in the body.

41. The Genetic Engineering of Plants Is Easier Than That of Animals • Plant advantages: • 1) they are easier to clone than animal cells; • 2) they can be grown in vast fields which allows massive production of desired products; • 3) they have the potential to be extremely lucrative. • ex: If the Flavor-Saver tomato becomes popular, the inventors will gain a virtual monopoly in the tomato market. • Molecular biologists can genetically engineer plants that can: • synthesize animal or plant proteins; • resist herbicides; • resist infection by plant viruses.

42. What Are the Environmental Risks of Recombinant DNA? • The long-term consequences are unknown. • Some argue that severe ecological effects will result. • For example, genetically engineered plants may eventually transfer their engineered genes into other plants. • Will pesticide resistant genes inserted into a crop plant be transferred to unrelated pest plants creating herbicide resistant weeds?

43. The Application of Recombinant DNA Technology Poses Moral Questions for Society • Diagnosis of genetic disease is far in advance of treatment. • Under such a situation, people may know that they have a genetic disease, but will not be able to do anything about it. • Will biologists try to modify genes that affect characteristics other than those responsible for disease? • Will future societies try to produce more intelligent citizens? • Will future societies try to produce fewer aggressive people?