1. DNA Technology & Genomics
Chapter 20
DNA Technology & Genomics

2. Biotechnology Terms Biotechnology Genetic Engineering Recombinant DNA
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 3 Steps 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 Agricultural applications
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