1. DNA Technology & Genomics
Chapter 6
Biotechnology:
DNA Technology & Genomics 1

2. The BIG 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?
Biotechnology
The use of living organisms (or substances from living organisms) for practical purposes 2

3. Biotechnology Use of organisms to create a desired product is not new
humans have been doing this for thousands of years
Alcohol fermentation (for brewing beer and food preservation)
INDIRECTLY manipulating DNA 3

4. Biotechnology Agriculture Plant and animal breeding
Selective breeding – altered the genomes of crops by breeding them with other plants.
INDIRECTLY manipulating DNA 4

5. Biotechnology today Genetic Engineering Direct manipulation of DNA
if you are going to engineer DNA & genes & organisms, then you need a set of tools to work with
this chapter is a survey of those tools… 5

6. Bioengineering Tool kit
Basic Tools
restriction enzymes
ligase
plasmids / cloning
DNA databases / probes
Advanced Tools
PCR
DNA sequencing
gel electrophoresis
Southern blotting
microarrays

7. Isolating DNA
Before DNA can be manipulated, it needs to be isolated from cells.
1. Cell membranes are disrupted
use a detergent
Salt used to neutralize lipids
2. DNA precipitation
ethanol used to dehydrate and aggregate DNA
Salt used to neutralize phosphate groups in DNA
3. DNA isolation / storage
e.g. Sharks do not get sick or get cancer. Scientists have determined the protein and the gene that is responsible for immunity in sharks.

8. DNA Isolation

9. 1. Cut DNA
To study the functions of individual genes, molecular biologists will
cut desired genes out of a genome
place the gene into bacterial plasmid
produce recombinant DNA

10. 1. Cut DNA treat isolated DNA with restriction enzymes
restriction endonucleases
Evolved in bacteria
Immune System
protection against viruses & other bacteria

11. Restriction enzymes - Action
cut DNA at specific sequences
Restriction (recognition) site
Are usually 4-8 bp in length
produces protruding ends
sticky ends that contain DNA nucleotides that lack complementary bases
Some do not produce protruding ends
blunt ends
CTGAATTCCG
GACTTAAGGC 
CTG|AATTCCG
GACTTAA|GGC  11

12. Restriction Enzymes Many different enzymes
named after organism they are found in
EcoRI, HindIII, BamHI, SmaI

13. Restriction Enzyme Cutting
sticky ends – enzyme digests (cuts) to make overhangs EcoRI 5’ G A A T T C 3’ 3’ C T T A A G 5’ 5’ G 3’ 5’ A A T T C 3’ 3’ C T T A A 5’ 3’ G 5’ 5’ overhang

14. Restriction Enzyme Cutting
PstI 5’ C T G C A G 3’ 3’ G A C G T C 5’ 5’ C T G C A 3’ 5’ G 3’ 3’ G 5’ 3’ A C G T C 5’ 3’ overhang

15. 2. Paste DNA Sticky ends allow: DNA Ligase
H bonds between complementary bases to anneal
DNA Ligase
enzyme “seals” strands
bonds sugar-phosphate backbone together
Condensation reaction 15

16. DNA Ligase T4 DNA ligase originated in T4 bacteriophages
used to chemically join two blunt ends of DNA together

17. 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. 17
Recombinant DNA molecule
GAATTC
CTTAAG

18. Exercise 1

19. Cut, Paste, Copy, Find… Word processing metaphor… 1. cut 2. paste
Isolate desired DNA
restriction enzymes
2. paste
ligase
3. copy
plasmids
bacteria
transformation
PCR
4. find
Southern blotting / probes 19

20. Remember… Prokaryotic genomes (e.g. bacteria) contain Chromosome
Plasmids
Chromosome
Plasmid

21. Plasmid pBR322 Plasmids Contain “accessory genes”
Antibiotic resistance
Can carry and express foreign genes
Plasmids are vectors
Vehicles by which DNA can be introduced into host cells

22. Plasmid Restriction Maps
Shows the location of cleavage sites for many different enzymes
These maps are used like road maps to the DNA molecule
Numbers beside restriction enzyme indicates at which base pair the DNA is cut by that particular enzyme

23. Sample Problem:
Determine the size and number of fragments that would be produced if the plasmid was digested with:
EcoRV and Pvu II?
2 cuts, therefore 2 fragments
Between sites:
=1879 bp
Rest:
=2482 bp

24. Why bacteria? Plasmid uptake is easy Cheap
Bacterium
Bacterial chromosome
Plasmid
Cell containing gene of interest
Recombinant DNA (plasmid)
Gene of interest
DNA of chromosome
Recombinate bacterium
Protein harvested
Basic research on protein
Copies of gene
Basic research on gene
Gene for pest resistance inserted into plants
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
Human growth hormone treats stunted growth
Protein expressed by gene of interest 3
Why bacteria?
Plasmid uptake is easy
Cheap
Reproduce rapidly and frequently
Produce multiple copies of the recombinant DNA and protein in a short amount of time
Recombinant DNA and proteins are kept in large storage database to be used later

25. Actin Protein Unnamed Protein
Protein Databases
Actin Protein Unnamed Protein

26. Review: Transformation vs. Recombination
the introduction of foreign DNA (usually a plasmid) into a bacterial cell
Recombination
Fragment of DNA composed of sequences originating from at least two different sources

27. 3. Copying (Cloning) Figure 20.4 TECHNIQUE
In this example, a human gene is inserted into a plasmid from E. coli. The plasmid contains the ampR gene, which makes E. coli cells resistant to the antibiotic ampicillin. It also contains the lacZ gene, which encodes -galactosidase. This enzyme hydrolyzes a molecular mimic of lactose (X-gal) to form a blue product. Only three plasmids and three human DNA fragments are shown, but millions of copies of the plasmid and a mixture of millions of different human DNA fragments would be present in the samples.
Sticky ends
Human DNA fragments
Human cell
Gene of interest
Bacterial cell
ampR gene
(ampicillin
resistance)
Bacterial plasmid
Restriction site
Recombinant DNA plasmids
lacZ gene (lactose breakdown)
Figure 20.4
3. Copying (Cloning) 1
Isolate plasmid DNA and human DNA. 2
Cut both DNA samples with the same restriction enzyme 3
Mix the DNAs; they join by base pairing. The products are recombinant plasmids and
many nonrecombinant plasmids.

28. Introduce the DNA into bacterial cells (transoformation).
RESULTS
Only a cell that took up a plasmid, which has the ampR gene, will reproduce and form a colony. Colonies with nonrecombinant plasmids will be blue, because they can hydrolyze X-gal. Colonies with recombinant plasmids, in which lacZ is disrupted, will be white, because they cannot hydrolyze X-gal. By screening the white colonies with a nucleic acid probe (see Figure 20.5), researchers can identify clones of bacterial cells carrying the gene of interest.
Colony carrying non- recombinant plasmid with intact lacZ gene
Bacterial clone
Colony carrying recombinant plasmid with disrupted lacZ gene
Recombinant bacteria 4
Introduce the DNA into bacterial cells (transoformation). 5
Plate the bacteria on agar containing
ampicillin and X-gal (lactose). Incubate until
colonies grow.

29. How do we know if it worked?
We know cloning worked if:
Transformation has occurred
i.e. there has been plasmid uptake into the bacteria
Recombination has occurred
i.e. foreign genes have been inserted into the bacterial plasmid

30. Selecting for successful transformation
Antibiotic resistance genes as a selectable marker
Plasmid has both “added” gene & antibiotic resistance gene
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) 30

31. Amp Selection If bacteria don’t pick up plasmid
they will not have antibiotic resistance
die on antibiotic (amp) plates
If bacteria pick up plasmid
survive on antibiotic (amp) plates
We have selected only those colonies that have undergone successful transformation

32. only transformed bacteria grow
Amp Selection
Ampicillin is the selecting agent
only transformed bacteria grow
all bacteria grow
Some bacteria on the LB/amp plate can have bacteria with recombinant plasmids and bacteria with non-recombinant plasmids
LB plate
LB/amp plate 32

33. Screening for successful recombination
Transformed colonies
have both recombinant and non-recombinant plasmids
recombinant
non-recombinant

34. LacZ gene codes for β galactosidase
LacZ Screening
LacZ gene codes for β galactosidase
Lactose (or X-gal) is our screening agent
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
Colour tells us to distinguish between 34

35. LacZ screening system We want these!!
Make sure inserted plasmid is recombinant plasmid
LacZ gene on plasmid produces digestive enzyme
lactose(X-gal)  blue
blue colonies
insert foreign DNA into LacZ gene breaks gene
lactose (X-gal)  blue
white colonies
white bacterial colonies have recombinant plasmid X X
We want these!! 35

36. Amp selection & LacZ screening
gene of interest
LacZ gene
- amp resistance
LB/amp
LB/amp/Xgal 36

37.     Cut, Paste, Copy, Find… Word processing metaphor… cut paste
restriction enzymes
paste
ligase
copy
plasmids
bacteria
transformation
PCR
find
Southern blotting / probes     37

38. 4. Find White colonies could have recombinant plasmids
desired and undesired genes
How do you find the conony with the gene of interest?

39. But how do we find colony with our gene of interest in it?
recombinant plasmids inserted into bacteria
gene of interest
human genome = 3 billion bases
fragments are cut to ~5000 bases
therefore ~ 600,000 fragments per cell.
But you have to use many cells to make sure you have a complete set in the library, so… you may have millions of cells that you extracted the DNA from, so… you would need millions of colonies, so the human genome cloned into bacteria would be a walk-in freezer full of petri dishes.
bacterial colonies (clones) grown on LB/amp/Xgal petri plates 39

40. Locating your gene of interest
DNA hybridization
find gene in bacterial colony using a probe
short, single stranded DNA molecule
complementary to part of gene of interest
tagged with radioactive P32 or fluorescence
heat treat genomic DNA
unwinds (denatures) strands
DNA hybridization between probe & denatured DNA 5’ 3’
labeled probe
genomic DNA G A T C

41. Hybridization 4 1 2 3 Locate Cloning expose film
locate colony on plate from film
Cloning
- plate with bacterial colonies carrying recombinant plasmids 1
plate
plate + filter
film 2
Replicate plate
press filter paper onto plate to take sample of cells from every colony
Hybridization
- heat filter paper to denature DNA
- wash filter paper with radioactive probe which will only attach to gene of interest
filter 3

42. Classwork/Homework Pg. 281 #1-3 Pg. 282 #6,7,10 Pg. 287 #16,18
Pg. 291 #2-7,10,11(b,c),15,16(a,b),17,19
Pg. 295 #4,5