1. DNA TECHNOLOGY
Chapter 20

2. BIOTECHNOLOGY
The manipulation of organisms or their components to perform tasks
Genetic Engineering – manipulation of genes
Gene cloning – making identical copies of a specific gene (a specific nucleotide sequence)

3. Techniques we will use in our labs
Transformation – putting a gene into bacteria (Luc gene into E. coli to make them glow)
Isolation of DNA – from cheek cells
PCR – used to amplify a section of DNA (taster gene now and later mtDNA)
Restriction Enzyme Digest – use of enzymes to cut DNA (plasmid mapping and taster gene)
Gel electrophoresis – used to separate different sizes of DNA fragments (plasmid mapping, taster gene, and later mtDNA)
Sequencing – determine exact base sequence of a section of DNA (later with mtDNA)

4. Figure 20.1 An overview of how bacterial plasmids are used to clone genes

5. OVERVIEW OF GENE CLONING
Isolation of plasmid DNA and gene to be cloned
Restriction enzyme digest
Gene inserted into plasmid
Plasmid put into bacteria
Cells cloned with gene as part of plasmid
Identification of desired clone
Various applications
Copies of protein product isolated
Copies of gene transferred to another cell

6. Cutting DNA
In nature, bacteria cut intruding (foreign) DNA with restriction enzymes
Enzymes cut at specific nucleotide sequences.
Recognize their own DNA by methyl (-CH3) groups added to adenines and cytosines
Restriction Site – specific sequence that is recognized by a specific restriction enzyme
Cuts the phosphodiester bonds (breaks backbone)
Most sites are symmetrical:
GAATTC
CTTAAG

7. Figure 20.2 Using a restriction enzyme and DNA ligase to make recombinant DNA

8. Most restriction enzymes cut in a staggered manner
G AATTC
CTTAA G
The ends are called “sticky” because they are complementary and would stick together
Additional DNA with same sticky ends (cut with same restriction enzyme) can be inserted.
Ligase added to make the needed phosphodiester bonds
GAATTC………..GAATTC
CTTAAG………..CTTAAG

9. Cloning vector – original plasmid used to carry foreign DNA into a cell and replicate there
Bacteria most commonly used host cells

10. Figure 20.3 Cloning a human gene in a bacterial plasmid: a closer look (Layer 3)

11. Identification of cells with clones of the right DNA gene done by using radioactive nucleic probes.
Probes are complements to part of the gene’s sequence
DNA must be denatured (H-bonds broken) for probe to have access to unwound DNA

12. Figure 20.4 Using a nucleic acid probe to identify a cloned gene

13. SOME PROBLEMS AND SOLUTIONS
Problem: expressing eukaryotic genes in prokaryotic cells
Solution: expression vector – cloning vector that contains the prokaryotic promoter just upstream of restriction site and inserted gene

14. Problem: Introns and noncoding regions in eukaryotes
Solution: use RNA and reverse transcriptase to make cDNA (complementary DNA or cDNA which has no introns)
Problem: differences between eukaryotes and prokaryotes
Solution: use yeast and some fungi

15. Figure 20.5 Making complementary DNA (cDNA) for a eukaryotic gene

16. Problem: Getting DNA into eukaryotic cells
Solutions: Electroporation – shock to make holes in cell membrane and microscopically thin needles

17. Figure 20.x2 Injecting DNA

18. PCR
Polymerase chain reaction – technique used to quickly copy (amplify) DNA and without cells
Used when DNA source is scarce or impure
Use heat resistant DNA polymerase, specific primers, and nucleotides to make many copies
Used to amplify a smaller section of DNA

19. Figure 20.7 The polymerase chain reaction (PCR)

20. GEL ELECTROPHORESIS
Gel electrophoresis – used to separate macromolecules based upon their size, charge, and other physical properties
DNA sample is cut with restriction enzymes
Different individuals produce different sized fragments which move through gel at different rates
Different alleles for same gene produce different sized fragments

21. GEL ELECTROPHORESIS
Restriction fragment length polymorphisms (RFLP) – differences in DNA sequences on homologous chromosomes that result in different restriction fragment patterns
RFLP’s used as genetic markers for making linkage maps (distance between genes)

22. Figure 20.8 Gel electrophoresis of macromolecules

23. Figure 20.9 Using restriction fragment patterns to distinguish DNA from different alleles

24. Figure 20.9 Using restriction fragment patterns to distinguish DNA from different alleles

25. SOUTHERN BLOTTING
Southern Blotting – hybridization technique used to determine presence of specific sequences within DNA
After gel electrophoresis is done, gel is blotted with nitrocellulose paper
DNA is transferred to paper in the banding pattern
Paper blot is exposed to radioactively labeled probes and then rinsed
Photographic film is laid over paper and radioactive probes that are attached to complements expose film

26. Figure 20.10 Restriction fragment analysis by Southern blotting

27. Figure 20.17 DNA fingerprints from a murder case

28. Figure 20.x1a Laboratory worker reviewing DNA band pattern

29. Figure 20.x1b DNA study in CDC laboratory

30. DNA SEQUENCING DNA sequencing – uses dideoxy chain termination
Each copied strand starts with same primer and ends with a dideoxyribonucleotide (ddNTP)
ddNTPs terminate strand because they do not have 3’ OH
Each ddNTP is fluorescently labeled differently (A, T, C, and G)
Labeled strands are sorted by size in a gel
Light source reads fluorescence, which determines base (A, T, C, or G)

31. DNA sequencing

32. DNA Microarrays
DNA microarrays – numerous amounts of small DNA strands of known sequences fixed to a glass slide or chip
Unknown DNA is labeled with fluorescent tags
Labeled DNA is washed over array and complementary strands bond and glow

33. DNA Microarrays

34. Cloning Cloning plants: single-cell cultures
Works because plant cells can de-differentiate
Cloning animals: Nuclear transplantation
First successful cloned mammal, Dolly, in 1997
Procedure does not have high success rate
Often clones have problems
Probably due to gene regulation differences in adult and embryo DNA

38. CC the cat (carbon copy); calico pattern different based on X inactivation
Cloned on 2002 at Texas A&M
Almost 10 years later CC, aka Copy Cat, is still in the College Station area. She has a mate, Smokey, and they live with their three offspring in a cat mansion built by Dr. Duane C. Kraemer, an A&M researcher who helped bring CC into the world.

39. Stem cells – relatively unspecialized cells that can both reproduce itself independently and, under appropriate conditions, differentiate into specialized cells
Found in embryos, skin, hair, eyes, dental pulp
Potentially used to treat many diseases like Alzheimer’s, Parkinson’s, Huntington’s etc.

41. APPLICATIONS OF BIOTECHNOLOGY
Gene therapy – replacing defective genes with a normal gene
In bone marrow cells, embryonic cells, and gametes
Problems:
Even if gene expressed, activity diminishes
Appropriate amounts expressed/ right time/ right place
Ex. SCID (severe combined immuno deficiency) lacks protein
Attempts to insert gene into bone marrow cells of 10 children worked in 9 patients, but caused leukemia in 2 (inserted into cell division gene)

42. Figure 20.16 One type of gene therapy procedure

43. Pharmaceutical products
Insulin
Human growth hormone
Tissue plasminogen activator (TPA)
Helps dissolve clots after heart attack/stroke
Make genetically engineered proteins to block or mimic cell receptors
Experimental drug that mimics receptor protein that HIV binds to so it attaches to drug instead of entering T-cells

44. Forensic Science – DNA fingerprinting
Environmental – using bacteria to clean up toxic wastes (extract heavy metals)
Agricultural – vaccines, antibodies, and growth hormones
Bovine growth hormone to make produce more milk
Genetically modified organisms (GMO)
Transgenic organisms – organisms that are given gene(s) from another species
Plants –genes for resistance to herbicides, less spoilage, larger fruits etc.
Medicine – using SNPs (single nucleotide polymorphisms) as markers; helps to identify disease causing alleles

46. Figure 20.19 Using the Ti plasmid as a vector for genetic engineering in plants

47. Figure 20.20 “Golden” rice contrasted with ordinary rice
Genes from daffodils and a bacterium that is inserted into rice plants so that rice can make beta-carotene

48. Sheep with gene for human blood protein
Figure “Pharm” animals
Sheep with gene for human blood protein
Protein is in sheep milk and isolated
Helps inhibit enzyme that destroys lungs in CF patients