Understanding DNA Technology and Genomics in Biotechnology

1. Chapter 20 DNA Technology & Genomics

2. Biotechnology Terms • Biotechnology • Process of manipulating organisms or their components to make useful products • Genetic engineering + tissue/cell culturing technologies • Genetic Engineering • Manipulation of individual genes or entire genomes • Insulin (insulin  E. coli bacteria OR yeast) & GMO (Genetically Modified Organism) • Recombinant DNA • Artificially created DNA • Typically, DNA is integrated from another species

3. Biotechnology Terms (Page 2) • Gene Cloning • Laboratory production of multiple copies of DNA segment • Therapeutic cloning – embryonic stem cells • Spinal cord injuries • Reproductive (organismal) cloning – Dolly the sheep • Restriction Enzymes • Enzymes that cut DNA at specific locations • Usually, derived from bacteria • Cut sites of DNA = restriction fragments • Sticky ends – restriction fragments usually have one end longer than the other

6. Quick Assignment • Relate the 6 terms just discussed in a concept map. • Be prepared to defend your arrangement

7. Cloning Process • 5 steps (first 2) 1. Identify & isolate the gene of interest • Involves finding a cloning vector – plasmid or organism used to carry the DNA sequence to be cloned 2. Cut gene of interest from original site & open up vector’s DNA using a ________ ________ • This ensures matching sticky ends on gene of interest & vector DNA

8. Cloning Process (Page 2) • 5 steps (3-4) 3. Combine the 2 DNA pieces (into a recombinant plasmid?) • Recombinant plasmid – plasmid + DNA fragments • Sealed together using DNA Ligase • Remember: we used ________ ________ to cut gene of interest from original site & cut vector’s DNA • This ensures matching sticky ends on gene of interest & vector DNA 4. Transfer the vector (recombinant plasmid) into a host cell • Usually involves bacterial transformation

9. Bacteria & Genetic Recombination • Conjugation • Bacterial Sex • Genetic material is exchanged by direct contact • Transduction • Phage transfer of DNA • Involves a phage vector • Phage moves the DNA from bacterium to other bacterium

10. Bacteria & Genetic Recombination • Transformation • Uptake of exogenous DNA • Griffith’s experiment - pathogenic DNA was transferred to benign bacteria • Most common method for genetic engineering

11. Step 5 • Select for transformed cells • Link the gene of interest with a reporter gene • Such as pBLU or pGLO • pBLU = Blue coloration • pGLO = fluorescent green under UV light • In Lab 6, we will insert the coloration gene and an ampicillin resistance gene to select for transformed cells

13. At this point… • You know which cells have the gene of interest • You can identify the cells that have the gene of interest • Now what? • You need to extract the gene of interest • How would you do that?

14. Nucleic Acid Hybridization • Detects the gene of interest • Uses a short, single stranded DNA or RNA called a nucleic acid probe • The nucleic acid probe is complementary to a known sequence in the gene of interest • Usually attach a radioactive isotope or fluorescent tag protein so that it is detectable

15. Genomic Libraries • Nucleic Acid Hybridization repeated many times produces a genomic library • Thousands of recombinant clones • Each has a piece of the original genome being studied

16. cDNA Library • cDNA = complementary DNA • mRNA is extracted from cells • Use what enzyme to make DNA from this mRNA? • Then make another strand of DNA using what enzyme? • cDNA library is only a portion of the genome • Portion that codes for mRNA • Exons? Introns? tRNA? rRNA?

17. Microarray Assay • Genome-wide study of gene expression • Different genes are in each well • Identifies gene interactions + provides clues to gene functions • Take samples throughout development + assay to determine which genes are expressed and at what stages • Detect patterns of expression throughout development • Detect likely response to a pathogenic agent

19. PCR • Polymerase Chain Reaction • Thermal cycling • Amplification of DNA • 3 Steps • Denaturation (Heating) • Annealing (Cooling) • Primer formation • Extension • DNA polymerase adds nucleotides at 3’ end

20. Gel Electrophoresis  DNA is negatively charged so it moves AWAY from the (-) cathode toward the (+) anode

22. Southern Blotting • Used to detect specific DNA sequences • Useful for comparing samples • Combines gel electrophoresis + nucleic acid hybridization

24. DNA Technology affects us… • Disease Diagnosis • PCR used to detect traces of viral DNA or RNA in sample • RFLP (Restriction Fragment Length Polymorphisms) • Different alleles have different RFLPs • Gene Therapy – alter afflicted genes • Pharmaceutical Production – Insulin production • Forensic Application – DNA fingerprints

25. Page 2 • Environmental cleanup • Genetically engineered microbes • Detoxification of specific wastes • Agricultural applications • Insert pest-resistant or drought-resistant genes • GMO (Genetically Modified Organisms) • You eat GMO corn, soybeans, canola and cottonseed oil • Probably at least weekly • 46% of GMOs are grown in US • Europe had 12 year moratorium on growing GE foods