1. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Chapter 10 Genetic Engineering: A Revolution in Molecular Biology 1

2. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 2 Genetic Engineering Basic knowledge is used to derive applied science or useful products Direct, deliberate modification of an organism’s genome –Bioengineering Biotechnology – use of an organism’s biochemical and metabolic pathways for industrial production

3. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 3 Tools and Techniques of Genetic Engineering Practical Properties of DNA Intrinsic properties of DNA hold true even in a test tube DNA heated from 90°C to 95°C; the two strands separate. The nucleotides can be identified, replicated, or transcribed. Slowly cooling the DNA allows complementary nucleotides to hydrogen bond and the DNA will regain double-stranded form Cooling Heating (a) DNA heating and cooling. DNA responds to heat by denaturing—losing its hydrogen bonding and thereby separating into its two strands. When cooled, the two strands rejoin at complementary regions. The two strands need not be from the same organisms as long as they have matching sites. Copyright © McGraw-Hill Education. Permission required for reproduction or display.

4. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 4 Enzymes for Dicing, Splicing, and Reversing Nucleic Acids Restriction endonucleases – recognize specific sequences of DNA and break phosphodiester bonds between adjacent nucleotides The enzymes can be used to cleave DNA at desired sites Recognize and clip the DNA at palindrome base sequences Used in the lab to cut DNA into smaller pieces – restriction fragments (b) Examples of endonucleases, palindromes and cutting patterns. The first two are staggered cuts, and the third is a blunt cut. Cutting pattern Endonuclease GAATTC CTTAAG EcoRI AAGCTT TTCGAA HindIII GGCC CCGG HaeIII Copyright © McGraw-Hill Education. Permission required for reproduction or display.

5. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 5 Restriction Fragment Length Polymorphisms DNA sequences vary, even among members of the same species Differences in the cutting pattern of specific restriction endonucleases give rise to fragments of differing lengths (RFLPs)

6. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 6 Enzymes for Dicing, Splicing, and Reversing Nucleic Acids Ligase – rejoins phosphate-sugar bonds (sticky ends) cut by endonucleases Used for final splicing of genes into plasmids and chromosomes

7. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 7 DNA Organism 1 (2) AGC T T TCGA AGCT TCGA AGCT DNA Organism 2 Action of restriction endonucleases. (1) A restriction endonuclease recognizes and cleaves DNA at the site of a specific palindromic sequence. Cleavage usually produces staggered tails called sticky ends that accept complementary tails for gene splicing. This palindrome is cut by Aci I. (2) The sticky ends can be used to join DNA from different organisms by cutting it with the same restriction enzyme, ensuring that both fragments have two complementary ends. This palindrome is cut by Taq I. CGA T T Restriction endonuclease makes staggered cut at palindrome. Site of cut Sticky ends C T A G G A T C C T A G G A T C (1) Copyright © McGraw-Hill Education. Permission required for reproduction or display.

8. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 8 Enzymes for Dicing, Splicing, and Reversing Nucleic Acids Reverse transcriptase – makes a DNA copy of RNA – cDNA cDNA can be made from mRNA, tRNA, or rRNA Provides a means of synthesizing eukaryotic genes from mRNA transcripts – synthesized gene is free of introns

9. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 9

10. 10 Methods for Analysis of DNA Gel electrophoresis - separates DNA fragments based on size –DNA samples are placed on soft agar gel and subjected to an electric current

11. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 11 Methods for Analysis of DNA –Negative charge of molecule causes DNA to move toward positive pole –Rate of movement is dependent on size of fragment – larger fragments move more slowly

12. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 12 Methods for Analysis of DNA –Fragments are stained for observation –Useful in characterizing DNA fragments and comparing for genetic similarities Well 12 5160 345 No. of base pairs in band 5035 4910 3160 2910 2760 2260 1510 1260 1010 Result, following development © Kathy Park Talaro Sample Known DNA Size Markers Copyright © McGraw-Hill Education. Permission required for reproduction or display. (b)

13. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Concept Check: DNA fragments move through the gel during electrophoresis according to their A.Size B.Charge C.Sequence D.Orientation 13

14. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Concept Check: DNA fragments move through the gel during electrophoresis according to their A.Size B.Charge C.Sequence D.Orientation 14

15. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 15 Methods for Analysis of DNA Nucleic acid hybridization and probes Single-stranded DNA can unite with other single-stranded DNA or RNA, and RNA can unite with other RNA – hybridization Foundation for gene probes – short DNA fragments of a known sequence that will base-pair with a stretch of DNA with a complementary sequence, if one exists in the sample Useful in detecting specific nucleotide sequences in unknown samples –Southern blot method – DNA fragments are separated by electrophoresis, denatured, and then incubated with DNA probes. Probes will attach to a complementary segment if present. –Isolate fragments from a mix of fragments and find specific gene sequences

16. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 16 Fluorescent in situ hybridization (FISH) - probes are applied to intact cells and observed for the presence and location of specific sequences –Useful for diagnostics or locating genes on a chromosome Methods for Analysis of DNA

17. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 17 Methods Used to Size, Synthesize, and Sequence DNA DNA sequencing – determining the actual order and type of bases for all types of DNA Most common sequencing technique is Sanger technique –Test strands are denatured to serve as a template to synthesize complementary strands –Fragments are divided into tubes that contain primers, DNA polymerase, all 4 nucleotides, and fluorescent labeled dideoxynucleotide

18. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 18 Sanger Sequencing Method

19. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 19 Methods Used to Size, Synthesize, and Sequence DNA Studying the genome of an organism has spawned new fields in biology: –Genomics - the systematic study of an organism’s genes and their functions –Proteomics - the study of an organism’s complement of proteins and functions mediated by the proteins –Metagenomics - the study of all the genomes in a particular ecological niche, as opposed to individual genomes from single species –Metabolomics - the study of the complete complement of small chemicals present in a cell at any given time

20. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 20 Methods Used to Size, Synthesize, and Sequence DNA Polymerase Chain Reaction (PCR) – method to amplify DNA; rapidly increases the amount of DNA in a sample –Primers of known sequence are added, to indicate where amplification will begin, along with special heat tolerant DNA polymerase and nucleotides –Repetitively cycled through denaturation, priming, and extension –Each subsequent cycle doubles the number of copies for analysis –Essentially important in gene mapping, the study of genetic defects and cancer, forensics, taxonomy, and evolutionary studies

21. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 21 Polymerase Chain Reaction (a)In cycle 1, the DNA to be amplified is denatured, primed, and replicated by a polymerase that can function at high temperature. The two resulting strands then serve as templates for a second cycle of denaturation, priming, and synthesis. 3′ 5′ 3′ 5′ DNA Sample Cycle 1 Denaturation Priming New strand Original strands Heat to 948C 728C Cycle 2 Cycles 3, 4,... repeat same steps, with the copies of DNA New strand Extension Cycle 2 Completed copies 4 copies 508C–658C 3′ 5′ 3′ 5′ Heat to 948C 508C to 658C Denaturation Oligonucleotide primers attach at ends of strands to promote replication of amplicons. 3′5′ Amplicons Primer 5′3′ 5′ Extension Heat-stable DNA polymerase synthesizes complementary strand. Cycle 1 Segment of DNA to be amplified Polymerase Strands separate. 728C 5′ 3′ 5′ 3′ 5′ 3′ Priming Copyright © McGraw-Hill Education. Permission required for reproduction or display. A view of the process after 6 cycles, with 64 copies of amplified DNA. Continuing this process for 20 to 40 cycles can produce millions of identical DNA molecules. (b) 1* fragment 2 copies 4 copies 8 copies 16 copies 32 copies 64 copies

22. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 22 Methods in Recombinant DNA Technology Recombinant DNA technology – the intentional removal of genetic material from one organism and combining it with that of a different organism –Objective of recombinant technology is cloning which requires that the desired donor gene be selected, excised by restriction endonucleases, and isolated –The gene is inserted into a vector (plasmid, virus) that will insert the DNA into a cloning host –Cloning host is usually bacterium or yeast that can replicate the gene and translate it into a protein product

23. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 23 Recombinant DNA Technique

24. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 24 Characteristics of Cloning Vectors Must be capable of carrying a significant piece of donor DNA Must be readily accepted by the cloning host Plasmids – small, well characterized, easy to manipulate and can be transferred into appropriate host cells through transformation Bacteriophages – have the natural ability to inject their DNA into bacterial hosts through transduction

25. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 25 Vector Considerations Origin of replication is needed so it will be replicated Vector must accept DNA of the desired size Gene which confers drug resistance to their cloning host

26. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 26 Desirable Features in a Cloning Host

27. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 27 Construction of a Recombinant, Insertion, and Genetic Expression Prepare the isolated genes for splicing into a vector by digesting the gene and the plasmid with the same restriction endonuclease enzymes creating complementary sticky ends on both the vector and insert DNA.

28. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 28 Construction of a Recombinant, Insertion, and Genetic Expression The gene and plasmid are placed together, their free ends base-pair, and ligase joins them The gene and plasmid combination is a recombination

29. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 29 Construction of a Recombinant, Insertion, and Genetic Expression The recombinant is introduced into a cloning host The cloning host transcribes the gene and translates the protein

30. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 30 Screening for Recombinant Microbes Use selective media to quickly identify recombinants Bacteria lack plasmid Bacteria carrying plasmid Regular nonselective medium with two types of colonies (1)(2) Culture of cloning host after incubation with recombinant plasmid Pure culture of bacteria containing cloned gene Selective medium with ampicillin Bacteria with recombinant plasmid Ampicillin- resistance gene Copyright © McGraw-Hill Education. Permission required for reproduction or display.

31. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Concept Check: Which of the following is a primary participant in cloning an isolated gene? A.Restriction Endonuclease B.Host Organism C.Vector D.Ligase E.All of the above 31

32. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Concept Check: Which of the following is a primary participant in cloning an isolated gene? A.Restriction Endonuclease B.Host Organism C.Vector D.Ligase E.All of the above 32

33. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 33 Biochemical Products of Recombinant DNA Technology Enables large scale manufacturing of life- saving hormones, enzymes, vaccines –Insulin for diabetes –Human growth hormone for dwarfism –Erythropoietin for anemia –Factor VIII for hemophilia –HBV vaccine

34. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 34 Recombinant Technology Examples

35. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 35 Genetically Modified Organisms (GMO, transgenic) Recombinant microbes –Pseudomonas syringae – prevents ice crystals –Bacillus thuringienisis – encodes an insecticide Many enzymes, hormones, and antibodies used in drug therapy are manufactured using mammalian cell culture –Cell cultures can modify the proteins Microbes to bioremediate disturbed environments Oncolytic adenoviruses – host range consists of cells that produce cancer-specific proteins

36. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 36 Transgenic Plants –Agrobacterium tumefaciens: a natural tumor-producing bacterium Ti plasmid inserts into the genomes of the infected plant cells Bacterium with selected gene Chromosome The large plasmid (Ti) of this bacterium can be used as a cloning vector for foreign genes that code for herbicide or disease resistance. The recombinant plasmids are taken up by the Agrobacterium cells, wihich multiply and copy the foreign gene. Genetically engineered Agrobacterium is inoculated into a culture of target plant cells and infects the cells. Bacterium fuses with the plant cell wall and the Ti plasmid enters. The T-DNA carries the herbicide gene into the plant chromosome. Mature plants can be grown from single cells, and these transgenic plants will express the new gene. Because the gene will be part of the plant’s genome, it will be transmitted to offspring in seeds. Isolated gene for herbicide resistance Ti plasmid T- DNA Agrobacterium cell Gene splicaed into Ti plasmid Recombirium Agrobacterium Agrobacterium with Ti plasmid vector Plant cell Process in plant (a) (b) (c) (d) (e) Copyright © McGraw-Hill Education. Permission required for reproduction or display.

37. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 37 Examples of Transgenic Plants

38. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 38 Transgenic Animals Use a virus to transfect a fertilized egg or early embryo Transgenic animals will transcribe and translate eukaryotic genes

39. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 39 Transgenic Animals as Disease Models Animal models have been designed to study human genetic diseases –Mouse models for CF, Alzheimer’s, sickle cell anemia –Sheep or goats manufacture proteins and excrete them

40. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 40 Examples of Transgenic Animals

41. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Genetic Treatments: Introducing DNA into the Body Gene therapy: correct or repair a faulty gene in humans –Ex vivo therapy: normal gene cloned in vectors, tissue removed from the patient –In vivo therapy: naked DNA or vector is directly introduced into the patient’s tissues 41 (1)Normal gene is isolated from healthy subject. Gene is cloned. Gene is inserted into retrovirus vecto Bone marrow sample is taken from patient with genetic defect. Stem cells from marrow are infected with retrovirus, (enlarged view) Transfected cells (red dots) are infused into patient. Patient is observed for expression of normal gene. Marrow stem cell 1 2 3 4 5 6 7 1 2 3 7 6 5 4 Copyright © McGraw-Hill Education. Permission required for reproduction or display

42. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 42 Genome Analysis DNA Fingerprinting – Every individual has a unique sequence of DNA Methods used include restriction endonucleases, electrophoresis, hybridization, and Southern blot

43. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 43 1 2 3 4 Probes for specific sequences added 1 2 3 4 Lanes Samples DNA 1 2 3 4 Visualized BandsPosition of Migration (a)Cells from different samples are processed to isolate their DNA. The DNA samples are exposed to endonucleases which snip them at specific sites into a series of different fragments. (b) Electrophoresis of DNA fragments separates them by size, with larger fragments nearer the wells. Although invisible to the unaided eye at this point, each lane contains a number of individual bands. Specific DNA sequences are identified by hybridization with fluorescently labeled nucleic acid probes or radioactive probes. (c) An actual DNA fingerprint used in a rape trial. Control lanes with known markers are in lanes 1, 5, 8, and 9. The second lane contains a sample of DNA from the victim’ blood. Evidence samples 1 and 2 (lanes 3 and 4) contain semen samples taken from the victim. Suspects 1 and 2 (lanes 6 and 7) were tested Can you tell by comparing evidence and suspect lanes which individual committed the rape? Marker PST Control Suspect 2 Suspect 1 Marker Evidence 2 Evidence 1 Victim Marker © DR. Michael Baird FORENSICTEST 1 2 3 4 5 6 7 8 9 Copyright © McGraw-Hill Education. Permission required for reproduction or display.

44. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 44 Genome Analysis Types of analysis –SNP – single nucleotide polymorphism –Markers VNTRs Microsatellite polymorphisms

45. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 45 Genome Analysis DNA Fingerprinting is used to Identify hereditary relationships Study inheritance of patterns of diseases Study human evolution Identify criminals or victims of disaster Analysis of mitochondrial DNA is used to trace evolutionary origins

46. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. 46 Genome Analysis Microarray analysis – track the expression of thousands of genes; used to identify and devise treatments for diseases based on the genetic profile of the disease

47. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Concept Check: If you wanted to identify which person licked a stamp, the best method to use would be A.Microarray Analysis B.DNA Fingerprinting C.Gene Therapy D.Anti-sense RNA Gene Silencing E.Any of the above would work 47

48. Copyright © McGraw-Hill Education. All rights reserved. No reproduction or distribution without the prior written consent of McGraw-Hill Education. Concept Check: If you wanted to identify which person licked a stamp, the best method to use would be A.Microarray Analysis B.DNA Fingerprinting C.Gene Therapy D.Anti-sense RNA Gene Silencing E.Any of the above would work 48