1. What do you notice about these phrases?
radar
racecar
Madam I’m Adam
Able was I ere I saw Elba
a man, a plan, a canal, Panama
Was it a bar or a bat I saw?

2. DNA Technology & Genomics
Chapter 20.
Biotechnology:
DNA Technology & Genomics
By Kim Foglia, Division Ave. H.S., Levittown, N..Y.
Modified by K. Crawford, Science Hill High School, Johnson City, TN

3. I. The Essential Questions…
How can we use our knowledge of DNA to:
diagnose disease or defect?
cure disease or defect?
change/improve organisms?
What are the techniques & applications of biotechnology?
direct manipulation of genes for practical purposes

4. One example: Glo Pigs! http://www.sciencebuzz.org/blog/when_pigs_glow

5. Genetically modified pigs glow under black light

6. Production of clotting factor IX in sheep’s milk
Ewe is given hormones to super-ovulate
Eggs are inseminated, harvested, then injected with rDNA.
Surrogate has transgenic egg implanted, baby lambs are born
When mature, they make milk
Clotting factor in milk must be isolated and purified for human use
Topic 4.4.9

7. Production of retinol and beta carotene by rice
Rice does not normally produce retinol or beta carotene
Rice has the precursor molecule to make retinol but not the enzyme
Retinol is necessary to prevent blindness
Topic 4.4.9

8. Production of retinol and beta carotene by rice
Gene isolated from daffodils and Erwinia bacteria is transferred to rice
Transgenic rice is then cross-bred with local rice strains
Voila! Rice will now produce retinol and beta-carotene
Topic 4.4.9

9. II. Historical perspective on manipulation of genes
A.Genetic manipulation of organisms is not new.
Humans have been doing this for thousands of years
plant & animal breeding
INDIRECTLY manipulating DNA

10. B. Evolution & breeding of food plants
Evolution of Zea mays from ancestral teosinte (left) to modern corn (right). The middle figure shows possible hybrids of teosinte & early corn varieties

11. “Descendants” of the wild mustard
Brassica sp.
What vegetables to you see descending from this plant?

12.
5.4.2

13. C. Animal husbandry / Animal breeding Allowing only certain plants and animals to breed.

14. Biotechnology today D. Genetic engineering involves: Our tool kit…
manipulation of DNA
If you are going to engineer DNA & genes & organisms, then you need a set of tools to work with.
This unit is a survey of those tools…
Our tool kit…

15. III. Bioengineering Tool kit
A. Basic Tools
restriction enzymes
ligase
plasmids / cloning
DNA libraries / probes
B. Advanced Tools
PCR
DNA sequencing
gel electrophoresis
Southern blotting
microarrays

16. C. Cut, Paste, Copy, Find… biotechnology terms.…
Word processing terms and analogous
biotechnology terms.…
Cut = restriction enzymes
Paste = ligase
Copy = plasmids
From bacteria
Process called transformation
Often requires use of PCR
Find = Southern blotting / probes

17. D. Cut DNA Restriction enzymes used
Also called restriction endonucleases
Discovered in 1960s
Evolved in bacteria to cut up foreign DNA (“restriction”)
Served as a protection against viruses & other bacteria
Bacteria protect their own DNA by methylation & by not using the base sequences recognized by the enzymes in their own DNA

18. E. Restriction enzymes Madam I’m Adam Action of enzyme
Cut DNA at specific sequences called restriction site
DNA bases form a symmetrical “palindrome”
Also produces protruding ends called sticky ends
Many different enzyme names.
Named after organism they are found in.
Examples: EcoRI, HindIII, BamHI, SmaI
CTGAATTCCG
GACTTAAGGC 
CTG|AATTCCG
GACTTAA|GGC 

19. Discovery of restriction enzymes
Werner Arber
Daniel Nathans
Hamilton O. Smith
Restriction enzymes are named for the organism they come from:
EcoRI = 1st restriction enzyme found in E. coli
Werner Arber discovered restriction enzymes. He postulated that these enzymes bind to DNA at specific sites containing recurring structural elements made up of specific basepair sequences.
Hamilton Smith verified Arber's hypothesis with a purified bacterial restriction enzyme and showed that this enzyme cuts DNA in the middle of a specific symmetrical sequence. Other restriction enzymes have similar properties, but different enzymes recognize different sequences. Ham Smith now works at Celera Genomics, the company who sequenced the human genome.
Dan Nathans pioneered the application of restriction enzymes to genetics. He demonstrated their use for the construction of genetic maps and developed and applied new methodology involving restriction enzymes to solve various problems in genetics.

20. F. Biotech use of restriction enzymes
GAATTC
CTTAAG
DNA
Restriction enzyme
cuts the DNA
Sticky ends (complementary
single-stranded DNA tails)
AATTC G
AATTC G G
CTTAA G
CTTAA
Add DNA from another source cut with same restriction enzyme
AATTC G G
AATTC
CTTAA G
DNA ligase
joins the strands.
Results in:
Recombinant DNA molecule
GAATTC
CTTAAG

21. G. Paste DNA Sticky ends allow: Ligase
H bonds between complementary bases to form
Ligase
enzyme “seals” strands
Bonds sugar-phosphate bonds
Is the covalent bond of DNA backbone

22. H. How to copy modified DNA
Use plasmids
which are small, self-replicating circular DNA molecules.
You insert DNA sequence into plasmid.
Plasmid serves as a vector = “vehicle” into organism; A way to get gene into the bacteria
Then conduct transformation:
Insert recombinant plasmid into bacteria
Bacteria will make lots of copies of plasmid.
Grow recombinant bacteria on agar plate
New bacterial colonies are clones (identical)
produces many copies of inserted gene

23. I. Recombinant plasmid contains:
Antibiotic resistance genes as a selectable marker
Restriction sites for splicing (adding) in gene of interest
Selectable marker
Plasmid has both “added” gene & antibiotic resistance gene
If bacteria don’t pick up plasmid then they die on antibiotic plates
If bacteria do pick up plasmid then they survive on antibiotic plates
Is selecting for successful transformation
selection
How do we know what’s the right combination of genes on a plasmid?
Trail and error research work.
selectable markers
high copy rate
convenient restriction sites
There are companies that still develop plasmids, patent them & sell them.
Biotech companies (ex. New England BioLabs)

24. Selection for plasmid uptake is first part of screening process.
Ampicillin becomes a selecting agent
Only bacteria with the plasmid will grow on amp plate
only transformed bacteria grow
all bacteria grow
LB plate
LB/amp plate

25. Need to screen the bacteria
Need to make sure bacteria have recombinant plasmid is second part of screening process.
plasmid
amp
resistance
LacZ gene
restriction sites
lactose  blue color
recombinant plasmid
amp
resistance
broken LacZ gene
lactose  white color X
inserted gene of interest
origin of replication
EcoRI
all in LacZ gene
BamHI
HindIII

26. LacZ is a screening system
Makes sure that the inserted plasmid is recombinant plasmid because not all plasmids have gene of interest.
LacZ gene on plasmid produces digestive enzyme
lactose(X-gal)  blue
Makes blue colonies
Insertion of foreign DNA into LacZ gene breaks gene
No enzyme produced
Makes white colonies
White bacterial colonies have recombinant plasmid X X
We want these!!

27. Amp selection & LacZ screening
gene of interest
LacZ gene
- amp resistance
LB/amp
LB/amp/Xgal

28. IV. What if you don’t have your gene conveniently on a chunk of DNA ready to insert into a plasmid?
Then you have to find your “gene of interest” out of the entire genome of the organism. How to do this?
By Kim Foglia, Division Ave. H.S., Levittown, N..Y.
Modified by K. Crawford, Science Hill High School, Johnson City, TN

29. A. Use DNA libraries
Cut up all of nuclear DNA from many cells of an organism
Use restriction enzyme
Clone all fragments into plasmids at same time
“shotgun” cloning
Create a stored collection of DNA fragments
Petri dish has a collection of all DNA fragments from the organism

30. B. Problems… A lot of junk! Introns! introns
human genomic library has more “junk” than genes in it
Introns!
If you want to insert a human gene into bacteria, you can’t have….
introns

31. Solution… Don’t start with DNA… Use mRNA
copy of the gene without the junk!
But in the end, you need DNA to insert into plasmid…
How do you go from RNA  DNA?
reverse transcriptase!

32. cDNA (copy DNA) libraries
Collection of only the coding sequences of expressed genes
Extract mRNA from cells
Use reverse transcriptase from retroviruses to make RNA  DNA
Insert into plasmid
Applications
Need edited DNA for expression in bacteria
Ex. - human insulin gene inserted into bacteria
Could you imagine how much that first insulin clone was worth to Genentech? One little piece of DNA in a plasmid worth billions! It put them on the map & built a multi-billion dollar biotech company.