Biotechnology Chapter 20

1
BiotechnologyChapter 20
2
DNA technology

• Sequencing manipulation of DNA
• Used in analyzing gene expression

3
DNA sequencing
4
Biotechnology

• Manipulation of organisms to make useful products

5
DNA Sequencing

• Nucleic acid hybridization
• Complementary base pairing
• One stand onto a different strand
• 2 techniques
• Dideoxy chain termination
• Next-generation sequencing

6
Dideoxy chain temination
DNA (template strand)
Primer
Deoxyribo-nucleotides
Dideoxyribonucleotides(fluorescently tagged)
Technique
3'
T G T T
5'
C T G AC T T C G A C A A
dATP
ddATP
5'
dCTP
ddCTP
DNA polymerase
dTTP
ddTTP
dGTP
ddGTP
P
P
P
P
P
P
G
G
3'
Labeled strands
DNA (template strand)
5'
3'
dd
G A C T G A
C T G AC T T C G A C A A
dd
A C T G A A G
dd
C T G A A G
dd
T G A A G
dd
G A A G
dd
A A G
dd
A G
A G C T G T T
dd
G C T G T T
3'
dd
C T G T T
C T G T T
C T G T T
C T G T T
C T G T T
C T G T T
C T G T T
5'
5'
3'
Shortest
Longest
Directionof movementof strands
Longest labeled strand
Detector
Laser
Shortest labeled strand
Results
Last nucleotideof longestlabeled strand
G A C T
G A A G C
Last nucleotideof shortestlabeled strand
7
Next generation
Technique
Genomic DNA is fragmented.
Results
A
4-mer
T
Each fragment is isolated witha bead.
G
3-mer
C
2-mer
Using PCR, 106 copies of eachfragment are made,
each attachedto the bead by 5' end.
1-mer
The bead is placed into a well withDNA
polymerases and primers.
Template strandof DNA
3'
5'
3'
5'
A
T
G
C
Primer
A solution of each of the four nucleotidesis
added to all wells and then washed off.The
entire process is then repeated.
G
G
G
A
T
C
A
T
C
A
T
C
A
T
C
G
Templatestrandof DNA
dCTP
dTTP
dGTP
dATP
C
PPi
A
A
A
A
PPi
DNApolymerase
Primer
If a nucleotide is notcomplementary to thenext
template base,no PPi is released, andno flash
of light is recorded.
The process is repeated until everyfragment has
a complete complementarystrand. The pattern of
flashes reveals thesequence.
If a nucleotide is joined to a growing strand,
PPi is released, causing a flash of light that
is recorded.
8
Genetic engineering

• Manipulation of genes
• Gene cloning
• Multiple copies of a single gene
• Produce a specific product

9
Fig. 20-2
Cell containing geneof interest
Bacterium
1
Gene inserted intoplasmid
Bacterialchromosome
Plasmid
Gene ofinterest
RecombinantDNA (plasmid)
DNA of chromosome
2
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in cultureto form a clone of
cellscontaining the clonedgene of interest
3
Gene ofInterest
Protein expressedby gene of interest
Copies of gene
Protein harvested
Basic research andvarious applications
4
Basicresearchon protein
Basicresearchon gene
Gene for pest resistance inserted into plants
Gene used to alter bacteria for cleaning up
toxic waste
Protein dissolvesblood clots in heartattack
therapy
Human growth hor-mone treats stuntedgrowth
10
Recombinant DNA

• 1970s
• Combining genes from different sources
• Even different species
• Combined into single DNA
• Example Bacteria mammal

11
Recombinant DNA

• Genetically modified bacteria
• Mass produce beneficial chemicals
• Insulin
• Growth hormone
• Cancer drugs
• Pesticides

12
Plasmid
13
Plasmid

• Small separate circular DNA
• Replicated same as main DNA
• Foreign DNA added to plasmid
• Replicated along with plasmid

14
Recombinant DNA

• Nucleases
• Enzymes that degrade DNA
• Restriction endonulceases
• Restriction enzymes
• Cut DNA into fragments
• Specific points

15
Recombinant DNA

• Restriction sites
• Where DNA is cut
• Restriction fragments
• Short DNA sequence

16
Recombinant DNA

• sticky ends
• Cuts in DNA sequences
• Single-stranded ends

17
Figure 20.6a
Bacterialplasmid
Restriction site
5'
3'
GAATTC
DNA
CTTAAG
5'
3'
Restriction enzyme cutsthe sugar-phosphatebackbo
nes at each arrow.
5'
3'
3'
5'
AATTC
G
CTTAA
G
5'
3'
5'
3'
Sticky end
18
Sticky ends
19
Recombinant DNA

• Insertion of DNA fragments from other sources
• Sticky ends
• Complementary match base pairs
• Hydrogen bonds
• DNA ligase
• Forms a phosphodiester bond

20
(No Transcript)
21
(No Transcript)
22
(No Transcript)
23
(No Transcript)
24
(No Transcript)
25
Recombinant DNA (Process)

• 1. Isolate gene of interest bacterial plasmid
• 2. Cut DNA plasmid into fragments
• 3. Mix DNA fragments with cut plasmid.
• Fragment with gene of interest is inserted into
  the plasmid
• 4. Recombinant plasmid is mixed with bacteria

26
Recombinant DNA (Process)

• 5. Bacteria with recombinant DNA reproduce
• 6. Isolate bacterial clones that contain gene of
  interest
• Producing protein of interest
• 7. Grow large quantities of bacteria that produce
  the protein

27
Recombinant DNA (Process)
28
Fig. 20-4-4
Hummingbird cell
TECHNIQUE
Bacterial cell
lacZ gene
Restrictionsite
Gene of interest
Stickyends
Bacterial plasmid
ampR gene
Hummingbird DNA fragments
Nonrecombinant plasmid
Recombinant plasmids
Bacteria carryingplasmids
RESULTS
Colony carrying recombinant plasmid with
disrupted lacZ gene
Colony carrying non-recombinant plasmidwith
intact lacZ gene
One of manybacterial clones
29
Recombinant DNA

• Vector
• DNA molecule-carries foreign DNA
• Enters replicates in the host
• Plasmids phages are common vectors
• Phages are larger than plasmid
• Can handle inserts up to 40 kilobases

30
PCR

• Polymerase chain reaction
• Amplify DNA
• Makes large quantities of DNA
• 1985

31
PCR

• Heated
• Denatured
• DNA primer
• Heat stable DNA polymerase
• Makes DNA

32
Fig. 20-8
5?
3?
TECHNIQUE
Targetsequence
3?
5?
Genomic DNA
1
3?
5?
Denaturation
5?
3?
2
Annealing
Cycle 1yields 2 molecules
Primers
3
Extension
Newnucleo-tides
Cycle 2yields 4 molecules
Cycle 3yields 8 molecules2 molecules(in
whiteboxes)match targetsequence
33
Gel electrophoresis

• Study DNA
• Polymer (gel)
• Restriction fragments
• Separates DNA based on charge size
• Nucleic acids negative charge (Phosphates)
• Migrate towards end (red)

34
Fig. 20-9
TECHNIQUE
Powersource
Mixture ofDNA mol-ecules ofdifferentsizes
Anode
Cathode


Gel
1
Powersource


Longermolecules
2
Shortermolecules
RESULTS
35
Fig. 20-10
Normal ?-globin allele
Normalallele
Sickle-cellallele
Large fragment
201 bp
175 bp
DdeI
DdeI
DdeI
Largefragment
DdeI
Sickle-cell mutant ?-globin allele
376 bp
201 bp175 bp
Large fragment
376 bp
DdeI
DdeI
DdeI
(b) Electrophoresis of restriction fragments
from normal and sickle-cell alleles
(a) DdeI restriction sites in normal and
sickle-cell alleles of ?-globin gene
36
Analyze gene expression

• cDNA
• Complementary DNA
• DNA made from an mRNA
• mRNA where gene is expressed
• RT-PCR
• Reverse transcriptase polymerase chain reaction

37
Analyze gene expression

• In vitro mutagenesis
• Cloned mutated gen
• Blocks expression
• RNAi
• RNA interference
• Nematodes, fruit fly

38
Analyze gene expression

• Genetic markers
• Detect abnormal disease
• SNP
• Single nucleotide polymorphisms
• Single base pair site where variation is found
• RFLP
• Restriction fragment length polymorphisms

39
Fig. 20-21
DNA
T
Normal allele
SNP
C
Disease-causingallele
40
Cloning

• Multicellular organisms come from a single cell.
• Offspring are identical

41
Cloning

• 1950
• Carrots
• Totipotent
• Mature cells-undifferentiated
• Give rise to any type of cells
• Common in plants

42
Cloning

• Nuclear transplantation
• Nucleus of an unfertilized/fertilized egg is
  removed
• Replaced with nucleus of differentiated cell
• Direct development of cell into tissues etc.

43
Cloning

• Removed nuclei from an egg
• Mammary cells
• Fused with egg cells
• Dolly, 1997, identical to mammary cell donor
• Died prematurely age 6
• Arthritis lung disease

44
Fig. 20-18
TECHNIQUE
Mammarycell donor
Egg celldonor
1
2
Egg cellfrom ovary
Nucleusremoved
Cells fused
Culturedmammary cells
3
3
Nucleus frommammary cell
Grown inculture
4
Early embryo
Implantedin uterusof a thirdsheep
5
Surrogatemother
Embryonicdevelopment
6
Lamb (Dolly)genetically identical tomammary
cell donor
RESULTS
45
(No Transcript)
46
Fig. 20-19
47
Cloning

• Few develop normally
• Abnormalities
• Epigenetic changes to the chromatin
• More methylation of chromatin
• Reprogram chromatin of differentiated cell

48
Stem cells

• Started 1998 at UW
• Early embryonic cells
• Potential to become any type of cell
• Master cell generates specialized cells
• Such as muscle cells, bone cells, or blood cells

49
Stem cells

• Embryos
• Bone marrow
• Umbilical cord blood
• Blood stem cells
• iPS
• Induced pluripotent stem cells
• Skin cells

50
Fig. 20-20
Adult stem cells
Embryonic stem cells
Early human embryoat blastocyst stage(mammalian
equiva-lent of blastula)
From bone marrowin this example
Cells generatingall embryoniccell types
Cells generatingsome cell types
Culturedstem cells
Differentcultureconditions
Differenttypes ofdifferentiatedcells
Blood cells
Nerve cells
Liver cells
51
Figure 20.21
Stem cell
Precursor cell
Experiment
Skinfibroblastcell
Oct3/4
Sox2
Four stem cell master regulatorgenes were
introduced, usingthe retroviral cloning vector.
c-Myc
Klf4
Induced pluripotentstem (iPS) cell
52
Medical applications

• Gene therapy
• Treat genetic defects
• Alters persons genes
• CF (vectors are viruses)
• SCID (immune disorder)
• Injected viral DNA with normal gene

53
Fig. 20-22
Clonedgene
Insert RNA version of normal alleleinto
retrovirus.
1
Viral RNA
Let retrovirus infect bone marrow cellsthat have
been removed from thepatient and cultured.
2
Retroviruscapsid
Viral DNA carrying the normalallele inserts into
chromosome.
3
Bonemarrowcell frompatient
Bonemarrow
Inject engineeredcells into patient.
4
54
Medical applications

• Transgenic animal
• Gene from one animal is inserted into another
• Goat milk protein anti-thrombin
• Isolated from milk
• pharm animals

55
Animals

• Transgenic animals engineered for specific traits
  
• Genetically create a racehorse
• Not have to breed
• Sheep with better wool??

56
Agricultural applications

• Manipulate tomatoes
• Do not ripen as fast
• Flavr-Savr
• Slows down ethylene production

57
Agricultural applications

• Introduce genes to plants
• Enable them to fix nitrogen
• Convert N2 to NH3
• Help eliminate use fertilizers

58
Agricultural applications

• Herbicide resistance
• Plant genetically resists the herbicide
• Insect resistance

59
Agricultural applications

• Transgenic rice
• golden rice
• Rice with genes that code for better absorption
  of iron and beta carotene
• First of many genetically engineered foods
• Helps dietary deficiencies

60
Forensics

• Genetic profile
• Individual genetic markers
• DNA fingerprint
• RFLP
• STR
• Short tandem repeats
• Occur in specific regions in genome
• Unique

61
Fig. 20-24
(a)
This photo shows EarlWashington just before his
release in 2001,after 17 years in prison.
Source of sample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim
17, 19
13, 16
12, 12
Earl Washington
16, 18
14, 15
11, 12
17, 19
13, 16
12, 12
Kenneth Tinsley
(b)
These and other STR data exonerated Washington
andled Tinsley to plead guilty to the murder.
62
Concerns over genetic engineering

• Genetically modified foods
• Harmful?
• Genetically engineered gametes
• Blonde and blue eyes??