BTG 403 Recombinant DNA Technology

1. BTG 403 Recombinant DNA Technology Dr. Friday N. Nwalo

2. Genetic Engineering • Genes are isolated, modified, and inserted into an organism • Made possible by recombinant technology • Cut DNA up and recombine pieces • Amplify modified pieces

3. Specificity of Cuts • Restriction enzymes cut DNA at a specific sequence • Number of cuts made in DNA will depend on number of times the “target” sequence occurs

4. Making Recombinant DNA 5’ G A A T T C 3’ C T T A A G one DNA fragment another DNA fragment 5’ G A A T T C 3’ 5’ C T T A A G 3’ In-text figurePage 254

5. Making Recombinant DNA nick 5’ G A A T T C 3’ 3’ C T T A A G 5’ nick DNA ligase action G A A T T C C T T A A G In-text figurePage 254

6. Using Plasmids • Plasmid is small circle of bacterial DNA • Foreign DNA can be inserted into plasmid • Forms recombinant plasmids • Plasmid is a cloning vector • Can deliver DNA into another cell

7. Using Plasmids DNA fragments + enzymes recombinant plasmids host cells containing recombinant plasmids Figure 16.4Page 255

8. Amplifying DNA • Fragments can be inserted into fast-growing microorganisms • Polymerase chain reaction (PCR)

9. Polymerase Chain Reaction • Sequence to be copied is heated • Primers are added and bind to ends of single strands • DNA polymerase uses free nucleotides to create complementary strands • Doubles number of copies of DNA

10. DNA heated to 90°– 94°C Primers added to base-pair with ends Mixture cooled; base-pairing of primers and ends of DNA strands DNA polymerases assemble new DNA strands Polymerase Chain Reaction Double-stranded DNA to copy Stepped Art Figure 16.6Page 256

11. Mixture cooled; base-pairing between primers and ends of single DNA strands DNA polymerase action again doubles number of identical DNA fragments Polymerase Chain Reaction Mixture heated again; makes all DNA fragments unwind Stepped Art Figure 16.6Page 256

12. DNA Fingerprints • Unique array of DNA fragments • Inherited from parents in Mendelian fashion • Even full siblings can be distinguished from one another by this technique

13. Tandem Repeats • Short regions of DNA that differ substantially among people • Many sites in genome where tandem repeats occur • Each person carries a unique combination of repeat numbers

14. Gel Electrophoresis • DNA is placed at one end of a gel • A current is applied to the gel • DNA molecules are negatively charged and move toward positive end of gel • Smaller molecules move faster than larger ones

15. Analyzing DNA Fingerprints • DNA is stained or made visible by use of a radioactive probe • Pattern of bands is used to: • Identify or rule out criminal suspects • Identify bodies • Determine paternity

16. Genome Sequencing • 1995 - Sequence of bacterium Haemophilus influenzae determined • Automated DNA sequencing now main method • Draft sequence of entire human genome determined in this way

17. Gene Libraries • Bacteria that contain different cloned DNA fragments • Genomic library • cDNA library

18. Endonucleases • type of enzyme in DNA strand.-produced nucleic acid strand breaks interior of nucleic acid strand.-restriction endonucleases-enzyme produced by bacteria that is used in recombinant DNA.-cuts open bacterial plasmid.-gene construct engineered to plasmid with ligasees. Plasmids back to bacterium.

19. Cloning Vectors • carrier for DNA during the recombinant DNA process.-plasmid-piece of free-floating DNA in the cytoplasm of bacteria.-double-stranded, circular molecules that replicate independently of the chromosome

20. TRANSFORMATION • Transformation- process of introducing free DNA into bacteria Competent cell- a cell that is capable of taking up DNA. Electroporation- The use of an electric shock to momentarily open or disrupt cell walls. Conjugation- the contact of bacteria that involves the exchange of DNA with a mating tube. Transformed cell- cell with new DNA Marker gene- a gene that identifies which organisms have been successfully transformed

21. AgroBacterium Transformation • Agro bacterium tumefacians is a bacterium that causes a disease known as crown gall in plants. • Infects plants by transferring its genetic material into plant cell. • Agrobacterium transformation is the most common technique for genetically engineered plants

22. Recombinant DNA Technology (genetic engineering)

23. Enzymes that cut and paste DNA Restriction enzymes cut DNA at specific base sequences called restriction sites

24. Enzymes that cut DNA are called restriction enzymes

25. Enzyme DNA ligase enzyme pastes cut ends back together

26. Cloning: the introduction of new or foreign genes into plasmids and other “vectors” This is when scientists take control of the natural processes that the bacteria have evolved to promote exchange of genes between individuals of the same or different “species” Circular extrachromosomal DNA found commonly in bacteria Plasmid DNA is replicated at same time chromosomal DNA is replicated Used to pass genes back and forth between different bacteria T G A C

27. Bacterial cells are efficient ways to produce lots of copies of a foreign gene introduced into a plasmid

28. Cloning • Plasmids serve as cloning vectors • “T”umor-”i”nducing • DNA (Ti plasmid) • contains 8 tumor- • inducing genes transformation • Use this plasmid to introduce a new gene into a plant chromosome

29. Review of molecular biologists’ toolbox • Plasmids • Restriction enzymes • DNA ligase • Host bacterial cells to replicate plasmids

30. Recombinant DNA technology has become a way for geneticists to express genes from other organisms in bacteria • Human insulin gene was cloned into a bacterial plasmid • and expressed (gene mRNA protein) in a bacterium in 1977. • Cheap and pure source of insulin • Humulin growth hormone was first recombinant • DNA product to be approved by FDA in 1992 • Currently over 100 products on market produced by • recombinant techniques

31. Mix plasmid and foreign DNA together with restriction enzyme and DNA ligase This plasmid has the lacZ gene inserted Section of foreign DNA with gene of interest Multiple cloning site inside lacZ gene (restriction site for insertion site for foreign gene) P O Plasmid cloning vector Foreign DNA Restriction site Restriction enzyme DNA ligase

32. Plasmid cloning vector Extra-chromosomal DNA carried by bacterial cell Ampicillin resistance gene ampR (selective marker) Multiple cloning site inside lacZ gene (restriction site for insertion site for foreign gene) Foreign gene inserted lacZ gene with promoter (used to switch in expression of foreign gene when inside a bacterial host cell)

33. Insert plasmid into host bacterial cell for replication Bacterial cell chromosome

34. Cultivate host cell to replicate and produce many copies of foreign gene Bacterial cell

35. Detecting cells that have foreign gene inserted in lacZ gene on plasmid • Need some way to check to see that foreign gene was inserted into the plasmid so when you cultivate the cell, you know you are producing more copies of foreign gene

36. Switching on expression of foreign gene during cultivation of host bacterial cell plasmid No foreign gene inserted RNA polymerase chromosome mRNA enzyme colored product If no foreign gene inserted into restriction site, then blue colored product is produced xGal (lactose)

37. If foreign gene is inserted into restriction site, then no colored product is produced RNA polymerase mRNA no enzyme xGal no product (no color)

38. Plating cells on agar surface to promote colony formation Visible colony of identical cells Medium contains ampicillin to allow only the bacterial cells that contain plasmid with ampR gene to grow Semisolid nutrient medium for bacterial cell to replicate to produce many daughter cells to form a visible colony

39. Cloning (restriction sites)

41. Types of vectors Maximum insert size (kilobases or kb [1000bp]) • Bacterial plasmid • bacteriophage • cosmids • bacterial artificial chromosome • yeast artificial chromosome 6-12 25 35 300 200-1000

42. Practical Features of DNA Cloning Vectors (Plasmids) • origin of replication (ori) • multiple cloning sites (MCS) or restriction sites • selectable markers • RNA polymerase promoter sequences • DNA sequencing primer sequences Allows bacteria with this plasmid to grow in presence of ampicillin antibiotic ori ampR lacZ gene MCS If plasmid picks up a foreign piece of DNA at the MCS, then the lacZ gene is non-functional

43. You can use plasmids to create a clone “library” Purpose: To distribute different sections of a DNA molecule or chromosome into a vector that allows the genes contained in the section to be characterized

44. Making a genomiclibrary Plate out to form colonies

45. Screening clones forplasmids that pickedup foreign DNA fragment

46. What if you know a part of the base sequence of the gene you are looking for? The Human Genome Project has given us this information for all the genes in our chromosomes

47. stopped

48. Polymerase chain reaction (PCR) • Has revolutionized molecular biology and biotechnology. • Most useful when you know at least some of the base sequence of the gene you are interested in • Only need to know a sequence containing 10-20 base pairs in a gene that may contain thousands of base pairs

49. Design primers that specifically target sequences at the ends of the foreign gene Plasmid 5’ 3’ Reverse primer T A T G C G Foreign gene C G C DNA polymerase Forward primer A T T C G C 3’ 5’

50. Polymerase Chain Reaction (PCR) Much more rapid approach to cloning than making or screening clone libraries. Makes lots of copies of foreign gene that is then inserted into plasmid Need to know part of sequence of gene