2. Phage DNA Donor cell Recipient cell A+A+ B+B+ A+A+ B+B+ A+A+ A+A+ B–B– A–A– B–B– A+A+ Recombinant cell Crossing over 1 2 3 4 5

3.  Genetic Engineering : process of manipulating genes and genomes  Biotechnology : process of manipulating organisms or their components for the purpose of making useful products. BioTechnology

4.  Recombinant DNA : DNA that has been artificially made, using DNA from different sources  eg. Human gene inserted into E.coli  Gene cloning : process by which scientists can product multiple copies of specific segments of DNA that they can then work with in the lab

5. Tools of Genetic Engineering  Restriction enzymes (restriction endonucleases): used to cut strands of DNA at specific locations (restriction sites)  Restriction Fragments: have at least 1 sticky end (single-stranded end)  DNA ligase : joins DNA fragments  Cloning vector : carries the DNA sequence to be cloned (eg. bacterial plasmid)

6. Using a restriction enzyme (RE) and DNA ligase to make recombinant DNA -ecoR1

7. Gene Cloning

8. Applications of Gene Cloning

9. 1) Isolate the gene of interest 2) Cut fragment DNA using restriction enzymes  restriction fragments 3) Insert the gene of interest into the plasmid 4) Close phosophodiester bonds with DNA ligase 5) Insert into vector (bacterium) by putting it in a chloride solution – Creates a permeable membrane

10. PART 2 – Inserting Hummingbird DNA into plasmid that contains lacZ 6) Redo process using a plasmid that already codes for ampicillin resistance (ampr gene) and lac Z – 7) Use restriction enzyme to cut plasmid and hummingbird DNA a) On the plasmid- Restriction enzyme must only be able to cut open Lac Z b) Hummingbird DNA is cut into pieces by R.E. 8) Grown on ampicillin laced Agar a) Only Ampicilling resistant colonies will grow (colonies that have taken up the plasmid b) Colonies that Lac Z gen was not cut will be blue c) Colonies that Lac Z gene was cut and a gene inserted will be white Animation 2

11. Figure 20.4 Bacterial plasmid TECHNIQUE RESULTS amp R gene lacZ gene Restriction site Hummingbird cell Sticky ends Gene of interest Humming- bird DNA fragments Recombinant plasmidsNonrecombinant plasmid Bacteria carrying plasmids Colony carrying non- recombinant plasmid with intact lacZ gene Colony carrying recombinant plasmid with disrupted lacZ gene One of many bacterial clones

12. Figure 20.4b RESULTS Bacteria carrying plasmids Colony carrying non- recombinant plasmid with intact lacZ gene Colony carrying recombinant plasmid with disrupted lacZ gene One of many bacterial clones

13. AP Biology Engineered plasmids Selectable marker  antibiotic resistance gene on plasmid  ampicillin resistance  selecting for successful transformation  successful uptake of recombinant plasmid plasmid amp resistance restriction sites EcoRI BamHI HindIII  Building custom plasmids  restriction enzyme sites  antibiotic resistance genes as a selectable marker ori

14. AP Biology Selection for plasmid uptake  Antibiotic becomes a selecting agent  only bacteria with the plasmid will grow on antibiotic (ampicillin) plate LB/amp plateLB plate all bacteria grow only transformed bacteria grow a a a a a a a a a a a a a a a cloning a a

15. AP Biology Need to screen plasmids  Need to make sure bacteria have recombinant plasmid plasmid amp resistance LacZ gene restriction sites lactose  blue color recombinant plasmid amp resistance broken LacZ gene inserted gene of interest all in LacZ gene EcoRI BamHI HindIII lactose  white color X origin of replication

16. AP Biology Screening for recombinant plasmid  Bacteria take up plasmid  Functional LacZ gene  Bacteria make blue color  Bacteria take up recombinant plasmid  Non-functional LacZ gene  Bacteria stay white color Which colonies do we want?

17. A genomic library is a collection of all of the cloned DNA fragments from a target genome  Genomic libraries can be constructed with different types of vectors  Plasmid library: genomic DNA is carried by plasmids  Phage library: genomic DNA is incorporated into bacteriophage DNA

19. Revere Transcriptase  Cells transcribe genes, process transcripts into mRNA  Research isolates mRNA, makes complimentary DNA transcripts using reverse transcriptase  Enzymes break down mRNA  DNA polymerase adds complementary bases to DNA   cDNA (resulting DNA) represents transcribed DNA

21. Techniques of Genetic Engineering  Transformation : bacteria takes up plasmid (w/gene of interest)  Nucleic acid hybridization : used to track gene of interest  PCR (Polymerase Chain Reaction): amplify (copy) piece of DNA without use of cells  Gel electrophoresis : used to separate DNA molecules on basis of size and charge using an electrical current (DNA  + pole)  Southern blotting : used to find a specific human gene  DNA microarray assays : study many genes at same time

22. PCR  Polymerase chain reaction  Makes multiple copies of a DNA sequence  Heat sample, separate DNA strands  Cool sample, primers bond  Heat-stable DNA polymerase builds complementary strands  Repeat

24. PCR (Polymerase Chain Reaction) : amplify (copy) piece of DNA without use of cells

25. Screening a Library for Clones Carrying a Gene of Interest  A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene  This process is called nucleic acid hybridization © 2011 Pearson Education, Inc.

26. A probe can be synthesized that is complementary to the gene of interest For example, if the desired gene is – Then we would synthesize this probe © 2011 Pearson Education, Inc. 5 3  CTCAT CACCGGC  5 3 G A G T A G T G G C C GG A G T A G T G G C C G

27. The DNA probe can be used to screen a large number of clones simultaneously for the gene of interest (remember genomic library) Once identified, the clone carrying the gene of interest can be cultured © 2011 Pearson Education, Inc.

28. Figure 20.7 Radioactively labeled probe molecules Gene of interest Probe DNA Single- stranded DNA from cell Film Location of DNA with the complementary sequence Nylon membrane Multiwell plates holding library clones TECHNIQUE 5 5 3 3 GAGTAGTGGCCG  CTCATCACCGGC 

29. Gel Electrophoresis  Gel: thin slab of thick jelly-like substance full of polymer fibers  DNA cut by restriction enzymes loaded into wells at one end  Gel hooked to power source (negative end is DNA end)  DNA has (-) charge (phosphates), so moved to (+) end  Bigger molecules move slower

30. Figure 20.9a Mixture of DNA mol- ecules of different sizes Power source Longer molecules Cathode Anode Wells Gel Shorter molecules TECHNIQUE 2     1

31. Figure 20.9b RESULTS

32.  In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis

33. RFLPs – restriction fragment length polymorphisms  Differences in restriction fragment lengths between two individuals  RFLP  Use restriction enzymes to cut up DNA  Unknown DNA; from suspects, victims, etc.  Compare length of restriction fragments  Rarely used anymore, but discovered numerous genetic disorders

34. STR Analysis Short tandem repeats –Unique number of repeats of the sequence –GATA repeated 12 times in one person, 35 times in another Thirteen sites throughout genome used DNA profiling usually uses this technique rather than specific sequence

37. SNP  Genetic markers called SNPs ( single nucleotide polymorphisms ) occur on average every 100–300 base pairs  Any SNP shared by people affected with a disorder but not among unaffected people may pinpoint the location of the disease-causing gene © 2011 Pearson Education, Inc.

38. Southern Blotting  A technique called Southern blotting combines gel electrophoresis of DNA fragments with nucleic acid hybridization  Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized on a “blot” of gel © 2011 Pearson Education, Inc.

39. Figure 20.11 DNA  restriction enzyme 3 2 14 TECHNIQUE I Normal  -globin allele II Sickle-cell allele III Heterozygote Restriction fragments Nitrocellulose membrane (blot) Heavy weight Gel Sponge Alkaline solution Paper towels II IIII II IIII II IIII Preparation of restriction fragments Gel electrophoresis DNA transfer (blotting) Radioactively labeled probe for  -globin gene Nitrocellulose blot Probe base-pairs with fragments Fragment from sickle-cell  -globin allele Fragment from normal  - globin allele Film over blot Hybridization with labeled probe Probe detection 5

40. Figure 20.10b Large fragment Normal allele Sickle-cell allele 201 bp 175 bp 376 bp (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles

41. Microarray Assay: used to study gene expression of many different genes Each well contains a DNA fragment for a specific gene

42. DNA Sequencing Relatively short DNA fragments can be sequenced by the dideoxy chain termination method, the first automated method to be employed Modified nucleotides called dideoxyribonucleotides (ddNTP) attach to synthesized DNA strands of different lengths Each type of ddNTP is tagged with a distinct fluorescent label that identifies the nucleotide at the end of each DNA fragment The DNA sequence can be read from the resulting spectrogram © 2011 Pearson Education, Inc.

43. Figure 20.12 DNA (template strand) TECHNIQUE 5 3 C C C C T T T G G A A A A G T T T DNA polymerase Primer 5 3 P P P OH G dATP dCTP dTTP dGTP Deoxyribonucleotides Dideoxyribonucleotides (fluorescently tagged) P P P H G ddATP ddCTP ddTTP ddGTP 5 3 C C C C T T T G G A A A A DNA (template strand) Labeled strands ShortestLongest 5 3 ddC ddG ddA ddG ddT ddC G T T T G T T T C G T T T C T T G G T T T C T G A G T T T C T G A A G T T T C T G A A G G T T T C T G A A G T G T T T C T G A A G T C G T T T C T G A A G T C A Direction of movement of strands Longest labeled strand Detector Laser Shortest labeled strand RESULTS Last nucleotide of longest labeled strand Last nucleotide of shortest labeled strand G G G A A A C C T

44. Genomics and Proteomics  Genomics  Study of whole genome  Thousands completed, others underway  Proteomics  Study of all protein products from genome  Many disorders result from misformed proteins  HUGE number of proteins for even small genomes

45. Applications of DNA Technology 1. Diagnosis of disease – identify alleles, viral DNA 2. Gene therapy – alter afflicted genes 3. Production of pharmaceuticals 4. Forensic applications – DNA profiling 5. Environmental cleanup – use microorganisms 6. Agricultural applications - GMOs

46. Gene therapy using a retroviral vector

48. Techniques of Genetic Engineering