Genetic Technology

1. Genetic Technology Manipulating Genes

2. A. Genetic Engineering • Genetic engineering (AKA recombinant DNA technology) is faster & more reliable method of selecting certain trait in population • Artificial selection is done by humans breeding specific individuals with certain traits - slow • Natural selection is nature selecting specific individuals with certain traits – very slow

3. Genetic engineering involves cleaving (cutting out) DNA from one organism into small fragments & inserting desired gene into host DNA of same or different species Desired gene Host DNA

4. Also called recombinant DNA technology since DNA gets recombined to make one new one • If plants or animals contain foreign DNA from this technology, are called transgenic organisms or genetically modified organism (GMO) • Example is tobacco plant that contains glowing gene from firefly – plant glows!!

5. Example of Transgenic Organisms Zebra Fish Firefly Bioluminescence Tobacco Plant Caterpillar

6. B. Steps of Engineering • Making transgenic or GMO takes 3 steps: 1. cleave DNA – isolate DNA fragment 2. vector - attach DNA fragment to carrier 3. insertion - insert DNA into host organism 3. Insert into host 2. Make a vector 1. Cleave DNA

7. DNA Cleavage • Must isolate small parts of DNA (DNA can contain millions of base pairs • use special enzymes called restriction enzymes that cut both sides of dsDNA at specific areas of nucleotide sequence • depending on which way DNA is cut, get 2 different ends: • sticky end • blunt end

8. sticky end • dsDNA is cut leaving some single strands • can only do that if there is a palindrome = letter order written same way backwards as forwards • ex: “mom” backwards is “mom” but with dsDNA, both sides are included • “GAATTC” on top side (forwards) and “CTTAAG” on bottom side (backwards) A T C C A G G A A T TC C A A G C T C T A G G T C C T T A A G G T T C G A G

9. restriction enzyme recognizes specific palindromes and will cut somewhere within there • ex: EcoRI recognizes GAATTC & will cut in b/t G – A on both sides leaving sticky ends ready to bond A T C C A G G A A T T C C A A G C T C T A G G T C C T T A A G G T T C G A G A A T T C C A A G C T C A T C C A G G G G T T C G A G T A G G T C C T T A A After Cleaving

10. TTAA and AATT sticky end have nothing to bond to, so if same restriction enzyme cuts DNA of organism and host’s organism, both sticky ends will match so bonding will be easier Vector Organism

11. Example of Sticky ends

12. blunt end • DNA is cut all way through like with scissors A T C C A G G A C T T C C A A G C T C T A G G T C C T G A A G G T T C G A G A T C C A G G A C T T C C A A G C T C T A G G T C C T G A A G G T T C G A G • both ends are bonded with other bases so are blunt After Cleaving

13. Examples of Restriction Enzymes

14. Attach to vector • Loose fragments of DNA need to be attached to vector (carrier) first • Two types of vectors: • Biological vector: bacteria plasmid or virus • Mechanical vector: micropipette or microscopic metal bullet

15. Since both DNA and vector were cleaved with same restriction enzyme, both ends will match • Join pieces using DNA ligase

16. Insertion into host • Recombined plasmid (or other vector) is inserted into host’s cell • When host replicates, inserted DNA also replicates producing more of that desired gene • bacterial plasmid can replicate every 20 min!

17. Bacterial plasmid • Inserts plasmid into bacteria’s cytoplasm • Virus • Injects DNA directly into host’s DNA • Process called transduction

18. Plasmid can replicate 500 times per cell, and each clone replicates 500 times… and so on • Clone: genetically identical copies of original Dolly (1996-2003) The first ever cloned animal

19. can also replicate DNA segments by using Polymerase Chain Reaction (PCR) • dsDNA strands are separated (unzipped) by heat • special heat-resistant enzymes replicate DNA • important advancement technique used to match DNA with very little DNA to begin with • Don’t need much DNA from crime scenes

20. PCR Technique

21. Example of Recombinant DNA

22. C. Uses for Genetic Engineering • recombinant DNA (genetic engineering) is currently useful in many areas of life • Industry • Medicine • Agriculture

23. Industry • clothing: bacteria E. coli are transgenic with DNA to make indigo dye • indigo dye in nature is VERY expensive, so can make blue jeans cheaply • food: making corn that has high protein content (corn is mostly carbohydrate) • fuel: use corn husks to make fuel for cars • sewage: clean water using bacteria

24. Medicine • hormone: can produce human growth hormone (hGH) to treat people with growth disorders (Achondroplasia, Turner syndrome) • medicine: produce human insulin (formerly bovine/cow) with bacterial plasmids • diseases: transgenic sheep are produced that produce Factor VIII protein for hemophiliacs • Vaccines: remove virus’ dangerous genes

25. Agriculture • making more/bigger/healthier/fresher food • Crops resistant to viruses and insects • canola plants make more canola oil • peanuts & soybeans that don’t cause allergic reactions • corn that can grow with very little water (survive drought)

26. D. The Human Genome Project • International effort started in1990, Human Genome Project (HGP) was organized to completely map and sequence human genome • complete sequence of nucleotides (3.2 billion) in human DNA (completed 2000). • Complete map of 20,000 genes (2006) on 23 sets of chromosomes

27. Human Genome Project Leaders of the Genome Project (Dr. Landers and Dr. Collins

28. How did they do that? • 1. How did they find out that genes U-Z are on chromosome set #2 and not on set #8? • 2. How do we know gene N is next to M and O and not somewhere else? • ANSWER: Linkage Maps A B C D A B C D QRS QRS U V W X Y Z U V W X Y Z Set# 2 Set# 8 LMNOP LMNOP

29. Linkage Map • Linkage map: genetic map that shows relative locations of genes on a chromosome • Found locations of genes on specific chromosomes, but didn’t know the order • Gene M is on chromosome 11 • Where on chromosome 11 is gene M? • Can find RELATIVE order of genes from a linkage map

30. Linkage Map Chromosome 11 Chromosome M

31. Using PCR, can make millions of copies of DNA fragments to find patterns in certain genes • Use genetic markers to trace inheritance of genes, which shows us where that gene is located relative to the others

32. Father has genes M & HD • Mother does not • Out of 5 kids, 3 inherited M, and of those 3, 2 also got HD • M & HD are close to each other

33. Gel Electrophoresis • Process of separating DNA fragments to compare sizes and therefore similarities • Electricity is sent through gel containing DNA fragments • DNA pieces will migrate toward bottom • Smaller pieces will “run” faster • Larger pieces will be stuck toward top large small

35. Genetic Markers

36. Paternity Tests Results: D2 not Dad’s Results: S2 adopted

37. How did they sequence it? • sequencing human genome compares DNA fragments to each other • pieces that overlap are pieced together All these fragments came from cleaving DNA into little workable pieces ABCD NOPQ DEFGHIJ ABCDEFGHIJKLMNOPQRSTUVWXYZ IJKLMNOP RSTUVWX WXYZ BCD LMNOPQRS By lining up pieces that overlap, can get entire sequence

38. E. Applications of HGP • Diagnosing genetic disorders – individuals find out if they are carrying gene for specific disease • Can be done for fetuses using epithelial cells (from amniotic fluid) • Dilemma – do YOU want to know if you have gene for cancer or heart disease? • Pros: can alter lifestyle NOW to help prevent cancer or heart disease from coming • Cons: always in fear about what may or may not happen

39. Gene therapy – inserting normal genes into human cells to correct genetic disorder • Cystic fibrosis, sickle-cell anemia, hemophilia, AIDS, cancer, heart disease are all being studied as genetic diseases in which gene therapy may work

40. Gene Therapy

41. DNA fingerprinting – compare unknown DNA to known DNA to find out if they match • DNA cut by restriction enzymes would show same sizes each time (same palindrome sequence) • Called restriction fragment length polymorphisms (RFLPs) • Solve crimes • Maternity/paternity UNKnown Known B A

42. DNA Fingerprinting

43. Paternity Tests Results: D2 not Dad’s Results: S2 adopted