1. Chapter 12
DNA technology

2. 12.1 Plasmids are used to customize bacteria
Recombinant DNA technology
Lab techniques used to combine genes from different sources (species) into a single DNA molecule
Use of natures “tools” have filled our recombinant DNA toolbox.
Enzymes, plasmids
Examples are plentiful
Bacteria produces human insulin, glow in dark bacteria, Flavor Saver Tomatoes, Golden Rice, Round-Up Ready Soybeans and Corn…

3. 12.1 Plasmids are used to customize bacteria
Introduction to a plasmid
Small circular DNA molecules that replicate separately from larger bacterial chromosomes
Used for “gene swapping” amongst bacteria
Antibiotic Resistance!!
Conjugation
“bacterial sex”

4. 12.1 Plasmids are used to customize bacteria
Plasmids (con’t)
After swapping sometimes genes are inserted into host chromosome which makes it a great vector
Gene carrier providing a means of integrating “genes” into a host

5. 12.1 Example of plasmids ability to transfer gene into host
Plant tumor caused from
Agrobacterium tumefaciens

6. 12.2 Enzymes used to cut and paste DNA
Restriction Enzyme – Enzymes that have the ability to cut DNA and is therefore used as molecular technology tool
Extracted from bacteria as they serve as a protective measure from invading viruses “restricting” them from surviving in cell.
Sticky ends – staggered (as opposed to a blunt cut) cuts in DNA exposing un-bonded nitrogen bases. Allows for recombinant DNA technology

7. 12.2 Enzymes used to cut and paste DNA
Restriction enzymes produce “sticky ends” which allow scientists to cut and paste
genes between organisms. They are like a molecular scissors.

8. 12.1 Example of plasmids ability to transfer gene into host
Tumor causing bacteria
Agrobacterium tumefaciens
Gene to be transferred
Cellular DNA
Inside plant cell, Agrobacterium inserts part of its DNA into host cell chromosome
Recombinant plasmid
Plant cell colonies
Transformed bacteria introduce plasmids into plant cells
Complete plant is generated from transformed cell
Agrobacterium tumefaciens is a bacteria that uses a plasmid to infect plants.
Scientists have utilized this plasmid to insert “genes of interest” into plants

9. 12.3 Plasmids can be cloned in recombinant plasmids
Recombinant DNA
Gene for human growth hormone
DNA recombination
Human Cell
Sticky ends
DNA insertion
Bacterial Cell
Bacterial chromosome
Bacterial cell for containing gene for human growth hormone
Plasmid
animation
review

10. 12.4 Cloned genes can be stored in genomic libraries (plasmid)
Genomic Library – The entire collection of all cloned DNA fragments from a genome
(genome – entire genes from one organism)
Uses
Storage, sequencing, study

11. 12.4 Cloned genes can be stored in genomic libraries (phages – virus)
Phage libraries much less space. 70 petri dishes can hold entire human genome
Compared to 300 petri dishes for plasmid library
Phage

12. 12.5 Reverse transcriptase helps make genes for cloning.
cDNA – Complementary DNA made
from expressed mRNA.
Made using reverse transcriptase from
retroviruses such as HIV. Advantage is
that it makes DNA from already edited
mRNA so DNA in our libraries won’t have
junk in them (saves space!)
Advantage of using cDNA
Studying specific cells and genes
associated with them. If we want to target
genes from a brain cell or a cancer cell
2. Smaller to work with.

13. Genomic Library vs. cDNA library

14. Genomic Library vs. cDNA library
Larger
Includes all possible genes from organism
Includes all exons and introns
cDNA
Smaller
Includes only expressed genes from specific cell
Includes only coding regions (exons)
Proteins can be expressed by transformed prokaryotes b/c no introns

15. 12.8 Nucleic acid probes identify clones carrying specific genes
How do we find books in the library?
Colonies will produce protein. If not…
Probing - Nucleic acid probe consists of a small synthesized strand of DNA, labeled with a radioactive isotope, that is complementary to the “gene of interest”

16. 12. 6 Recombinant cells and organisms
Bacteria
Plasmids make ideal vectors, grown rapidly and cheaply, “secretion” of proteins
Prokaryotic organisms not as good at producing eukaryotic proteins.
Yeast
Eukaryotic so better at producing eukaryotic proteins. Single celled, also has a plasmid
Mammals
Many proteins are glycoproteins unique to mammal protein synthesis
Difficult for secretion (via milk in some cases). Gene of interest is accompanied with a promotor specific to genes in mammary gland. SpiderGoat!

18. 12.7 DNA technology is changing pharmaceutical industry
Hormones
Insulin and HGH via recombinant E.coli
Diagnosis and Treatment of disease
Genetic Testing very common
Gene Therapy not where they hoped it would be
Vaccine – harmless variation of pathogen given to trigger immune response
Genetically engineer bacteria to make surface proteins associated with pathogen
Alter the gene of the actual pathogen that makes it harmful

19. 12.9 Microarray – measures gene expression
Collect Tissue
Isolate RNA - density
Isolate mRNA – poly A tails
Make labeled cDNA – using RT
Apply cDNA to microarray (hybridize)
Scan microarray
Analyze data
*Red = expressed in cancer
*Green = expressed in healthy
Yellow = expressed in both
(* genes of interest)
Analyze data

20. Lab Techniques to be familiar with
1. DNA microarray – Can be used to determined which genes in a cell are being expressed. Provides a large scale analysis of gene expression. Animation
2. Gel electrophoresis – Used to separate fragments of DNA (produced by restriction enzymes) to make DNA comparisons. Animation
3. PCR – Polymerase Chain Reaction. Uses heat resistant Taq enzyme to make billions of copies of targeted DNA sequences. Animation
4. CRISPR-Cas9 – Highly specific technique used to “target” specific place on DNA to alter. Animation

21. 12.10 Electrophoresis

22. 12.10 Electrophoresis

23. 12.10 Electrophoresis
Whose the father?

24. 12.14 PCR – Polymerase Chain Reaction
Use to amplify DNA (game changer)
Id of viruses (can determine if HIV has been integrated into host)
Amplify indiscriminate amount of DNA at a crime scene
Genetic testing
Technology
Primers – “paper clips” that locate and isolate the region of DNA to be copied (these are programmable – for example there are HIV primers). This is where DNA polymerase binds
Polymerase – copies DNA. Must be able to withstand temps required. Thermus aquaticus – bacteria found in thermal vents
Thermal cycler – machine that cycle temperatures needed
95 – breaks DNA 50 – allows DNA primers to bind – DNA is copied

25. animation
Polymerase binds to primers and copies DNA