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NOTES - CH 20: DNA Technology

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Title: Lecture #9 Date _____ Author: Chris Hilvert Last modified by: WLHS_Teacher Created Date: 11/3/2000 4:10:38 PM Document presentation format – PowerPoint PPT presentation

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Title: NOTES - CH 20: DNA Technology


1
NOTES - CH 20 DNA Technology
2
  • ? BIOTECHNOLOGY the use of living organisms or
    their components to do practical tasks
  • -microorganisms to make wine/cheese
  • -selective breeding of livestock
  • -production of antibiotics

3
  • Practical goal of biotech improvement of
    human health and food production

4
Recombinant DNA
  • ? Recombinant DNA DNA in which genes from 2
    different sources are linked
  • ? Genetic engineering direct manipulation of
    genes for practical purposes

5
  • Toolkit for DNA technology involves
  • -restriction enzymes
  • -DNA vectors
  • -host organisms

6
  • RESTRICTION ENZYMES
  • (a.k.a. ENDONUCLEASES)
  • enzymes that recognize
  • short, specific nucleotide
  • sequences called
  • restriction sites
  • ? in nature, these enzymes
  • protect bacteria from intruding
  • DNA they cut up the DNA
  • (restriction) very specific

7
Restriction Enzymes
  • ? restriction sites are symmetrical
    (palindromes) in that the same sequence of 4-8
    nucleotides is found on both strands, but run in
    opposite directions
  • ? restriction enzymes usually cut phosphodiester
    bonds of both strands in a staggered manner
    producing single stranded sticky ends

8
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9
Restriction Enzymes (cont.)
  • ? sticky ends of restriction fragments are used
    in the lab to join DNA pieces from different
    sources (complementary base pairing)
  • ? unions of different DNA sources can be made
    permanent by adding the enzyme DNA ligase

10
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12
  • CLONING VECTOR DNA molecule that can carry
    foreign DNA from test tubes back into cells
    replicate once there
  • -bacterial plasmids (small, circular DNA
    molecules that replicate within bacterial cells)
  • -viruses

13
  • HOST ORGANISMS
  • bacteria are commonly
  • used as hosts in genetic
  • engineering because
  • 1) DNA can easily be isolated from
    reintroduced into bacterial cells
  • 2) bacterial cultures grow quickly, rapidly
    replicating any foreign genes they carry.

14
Steps Involved in Cloning a Human Gene
  • 1)  Isolate human gene to clone
  • 2) Isolate plasmid from bacterial cell
  • 3) Add restriction endonuclease to cut out
    human gene add same R.E. to open up bacterial
    plasmid (creates the same sticky ends)
  • 4) Add human gene to the open bacterial
    plasmid and seal with DNA ligase

plasmid
Human gene
15
Cloning a Human Gene (cont.)
  • 5) Insert recombinant DNA plasmid back into
    bacterial cell
  • 6) As bacterial cell reproduces, it makes copies
    of the desired gene
  • 7) Identify cell clones carrying the gene of
    interest.
  • -HOW? Which ones took up the gene are making
    insulin?

16
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17
Bacterial plasmids in gene cloning
18
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19
DNA Analysis Genomics
  • ? PCR (polymerase chain reaction)
  • ? Gel electrophoresis
  • ? Restriction fragment analysis (RFLPs)
  • ? Southern blotting
  • ? DNA sequencing
  • ? Human genome project

20
The Polymerase Chain Reaction (PCR)
  • ? allows any piece of DNA to be quickly amplified
    (copied many times) in vitro.
  • ? DNA is incubated under
  • appropriate conditions
  • with special primers
  • DNA polymerase
  • molecules
  •  

21
PCR (continued) ? BILLIONS of copies of DNA are
produced in just a few hours (enough to use for
testing)
In 6 cycles of PCR cycle 1 2 copies cycle 2 4
copies cycle 3 8 copies cycle 4 16 copies cycle
5 32 copies cycle 6 64 copies cycle 20
1,048,576!!
22
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23
Polymerase Chain Reaction (PCR)
  • ? PCR is highly specific primers determine the
    sequence to be amplified
  • ? only tiny amounts of DNA are needed

Remember these?
24
Starting materials for PCR
  • ? DNA to be copied
  • ? Nucleotides
  • ? Primers
  • ? Taq polymerase
  • (DNA polymerase isolated from bacteria living in
    hot springstheir enzymes can withstand high
    temps!)

25
Steps of PCR
  • 1)  Heat to separate DNA strands (95ºC)
  • 2) Cool to allow primers to bind (55ºC)
  • 3) Heat slightly so that DNA polymerase extends
    the 3 end of each primer (72ºC)
  • 4) Repeat steps 1-3 many times!!!

26
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27
5?
3?
TECHNIQUE
Target sequence
Genomic DNA
5?
3?
Denaturation
5?
3?
5?
3?
Annealing
Cycle 1 yields 2 molecules
Primers
Extension
New nucleotides
Cycle 2 yields 4 molecules
Cycle 3 yields 8 molecules 2 molecules (in white
boxes) match target sequence
28
5?
3?
TECHNIQUE
Target sequence
Genomic DNA
5?
3?
29
Figure 20.8b
5?
3?
Denaturation
3?
5?
Annealing
Cycle 1 yields 2 molecules
Primers
Extension
New nucleo- tides
30
Figure 20.8c
Cycle 2 yields 4 molecules
31
Figure 20.8d
Cycle 3 yields 8 molecules 2 molecules (in white
boxes) match target sequence
32
Applications of PCR
  • ? DNA / forensic analysis of tiny amounts of
    tissue or semen found at crime scene
  • ? DNA from single embryonic cells for prenatal
    diagnosis
  • ? DNA or viral genes from cells infected with
    difficult-to-detect viruses such as HIV
  • ? used extensively in Human Genome Project to
    produce linkage maps without the need for large
    family pedigree analysis.

33
PCR works like a copying machine for DNA!
34
DNA Analysis
  • ? Gel electrophoresis separates nucleic acids
    or proteins on the basis of size or electrical
    charge creating DNA bands of the same length

35
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36
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37
Restriction fragment analysis
  • ? Restriction fragment length polymorphisms
    (RFLPs)
  • ? Southern blotting process that reveals
    sequences and the RFLPs in a DNA sequence
  • ? DNA Fingerprinting

38
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39
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40
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41
DNA Sequencing
  • ? Determination of nucleotide sequences (Sanger
    method, sequencing machine)
  • ? Human Genome Project

42
DNA Sequencing
  • ? Relatively short DNA fragments can be sequenced
    by the dideoxy chain termination method, the
    first automated method to be employed
  • ? Modified nucleotides called dideoxyribonucleotid
    es (ddNTP) attach to synthesized DNA strands of
    different lengths
  • ? Each type of ddNTP is tagged with a distinct
    fluorescent label that identifies the nucleotide
    at the end of each DNA fragment
  • ? The DNA sequence can be read from the resulting
    spectrogram

43
Figure 20.12
TECHNIQUE
Primer
Deoxyribonucleotides
Dideoxyribonucleotides (fluorescently tagged)
DNA (template strand)
3?
T
G
5?
C
T
dATP
ddATP
T
T
5?
G
dCTP
ddCTP
A
DNA polymerase
C
dTTP
ddTTP
T
dGTP
T
ddGTP
C
G
P
P
P
P
P
P
A
G
G
C
A
OH
H
3?
A
DNA (template strand)
Labeled strands
3?
5?
ddG
C
A
T
ddA
G
C
ddC
C
T
A
ddT
T
T
G
C
ddG
G
G
G
T
A
A
A
A
ddA
A
T
ddA
A
A
A
A
A
A
G
G
C
ddG
G
G
G
G
G
C
G
ddC
C
C
C
C
C
C
C
T
T
A
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
C
G
G
G
G
G
G
G
G
G
T
A
T
T
T
T
T
T
T
T
5?
T
3?
A
T
T
T
T
T
T
T
T
Shortest
Longest
Direction of movement of strands
Longest labeled strand
Detector
Laser
Shortest labeled strand
RESULTS
Last nucleotide of longest labeled strand
G
A
C
T
G
A
Last nucleotide of shortest labeled strand
A
G
C
44
Figure 20.12a
TECHNIQUE
Primer
Deoxyribonucleotides
Dideoxyribonucleotides (fluorescently tagged)
DNA (template strand)
3?
T
G
5?
C
dATP
T
ddATP
T
T
5?
G
dCTP
ddCTP
A
DNA polymerase
dTTP
C
ddTTP
T
dGTP
ddGTP
T
C
G
P
P
P
P
P
P
A
G
G
C
A
OH
H
A
3?
45
Figure 20.12b
TECHNIQUE (continued)
DNA (template strand)
Labeled strands
3?
5?
ddG
C
A
T
ddA
C
G
ddC
C
T
A
ddT
T
T
G
C
G
ddG
G
G
A
T
A
A
A
A
ddA
ddA
A
A
A
T
A
A
A
G
G
C
ddG
G
G
G
G
G
G
C
C
C
C
C
ddC
C
C
C
A
T
T
T
T
T
T
T
T
T
G
C
G
G
G
G
G
G
G
G
T
T
T
A
T
T
T
T
T
T
T
A
T
T
T
T
T
T
T
5?
T
3?
Shortest
Longest
Direction of movement of strands
Longest labeled strand
Detector
Laser
Shortest labeled strand
46
Figure 20.12c
Direction of movement of strands
Longest labeled strand
Detector
Laser
Shortest labeled strand
RESULTS
Last nucleotide of longest labeled strand
G
A
C
T
G
A
Last nucleotide of shortest labeled strand
A
G
C
47
Analyzing Gene Expression
  • ? Nucleic acid probes can hybridize with mRNAs
    transcribed from a gene
  • ? Probes can be used to identify where or when a
    gene is transcribed in an organism

48
  • ? In situ hybridization uses fluorescent dyes
    attached to probes to identify the location of
    specific mRNAs in place in the intact organism

49
Figure 20.14
50 ?m
50
Studying the Expression of Interacting Groups of
Genes
  • ? Automation has allowed scientists to measure
    the expression of thousands of genes at one time
    using DNA microarray assays
  • ? DNA microarray assays compare patterns of gene
    expression in different tissues, at different
    times, or under different conditions

51
Figure 20.15
TECHNIQUE
Isolate mRNA.
Tissue sample
Make cDNA by reverse transcription,
using fluorescently labeled nucleotides.
mRNA molecules
Labeled cDNA molecules (single strands)
Apply the cDNA mixture to a microarray, a
different gene in each spot. The cDNA
hybridizes with any complementary DNA on the
microarray.
DNA fragments representing a specific gene
DNA microarray
Rinse off excess cDNA scan microarray for
fluorescence. Each fluorescent spot (yellow)
represents a gene expressed in the tissue sample.
DNA microarray with 2,400 human genes
52
Figure 20.15a
DNA microarray with 2,400 human genes
53
Determining Gene Function
  • ? One way to determine function is to disable the
    gene and observe the consequences
  • ? Using in vitro mutagenesis, mutations are
    introduced into a cloned gene, altering or
    destroying its function
  • ? When the mutated gene is returned to the cell,
    the normal genes function might be determined by
    examining the mutants phenotype

54
  • ? Gene expression can also be silenced using RNA
    interference (RNAi)
  • ? Synthetic double-stranded RNA molecules
    matching the sequence of a particular gene are
    used to break down or block the genes mRNA

55
  • ? In humans, researchers analyze the genomes of
    many people with a certain genetic condition to
    try to find nucleotide changes specific to the
    condition
  • ? Genetic markers called SNPs (single nucleotide
    polymorphisms) occur on average every 100300
    base pairs
  • ? SNPs can be detected by PCR, and any SNP shared
    by people affected with a disorder but NOT among
    unaffected people may pinpoint the location of
    the disease-causing gene

56
Figure 20.16
DNA
T
Normal allele
SNP
C
Disease-causing allele
57
20.3 Cloning organisms may lead to production of
stem cells for research and other applications
  • ? Organismal cloning produces one or more
    organisms genetically identical to the parent
    that donated the single cell

58
Cloning Plants Single-Cell Cultures
  • ? A totipotent cell is one that can generate a
    complete new organism
  • ? Plant cloning is used extensively in agriculture

59
Figure 20.17
Cross section of carrot root
2-mg fragments
Single cells free in suspension began to divide.
Embryonic plant developed from a cultured single
cell.
Fragments were cultured in nu- trient
medium stirring caused single cells to shear off
into the liquid.
Plantlet was cultured on agar medium. Later it
was planted in soil.
Adult plant
60
Cloning Animals Nuclear Transplantation
  • ? In nuclear transplantation, the nucleus of an
    unfertilized egg cell or zygote is replaced with
    the nucleus of a differentiated cell
  • ? Experiments with frog embryos have shown that a
    transplanted nucleus can often support normal
    development of the egg
  • ? However, the older the donor nucleus, the lower
    the percentage of normally developing tadpoles

61
Figure 20.18
EXPERIMENT
Frog embryo
Frog egg cell
Frog tadpole
UV
Fully differ- entiated (intestinal) cell
Less differ- entiated cell
Donor nucleus trans- planted
Donor nucleus trans- planted
Enucleated egg cell
Egg with donor nucleus activated to
begin development
RESULTS
Most stop developing before tadpole stage.
Most develop into tadpoles.
62
Reproductive Cloning of Mammals
  • ? In 1997, Scottish researchers announced the
    birth of Dolly, a lamb cloned from an adult sheep
    by nuclear transplantation from a differentiated
    mammary cell
  • ? Dollys premature death in 2003, as well as her
    arthritis, led to speculation that her cells were
    not as healthy as those of a normal sheep,
    possibly reflecting incomplete reprogramming of
    the original transplanted nucleus

63
Figure 20.19
TECHNIQUE
Mammary cell donor
Egg cell donor
Egg cell from ovary
Nucleus removed
Cells fused
Cultured mammary cells
Nucleus from mammary cell
Grown in culture
Early embryo
Implanted in uterus of a third sheep
Surrogate mother
Embryonic development
Lamb (Dolly) genetically identical to mammary
cell donor
RESULTS
64
Figure 20.19a
TECHNIQUE
Mammary cell donor
Egg cell donor
Egg cell from ovary
Nucleus removed
Cells fused
Cultured mammary cells
Nucleus from mammary cell
65
Figure 20.19b
Nucleus from mammary cell
Grown in culture
Early embryo
Implanted in uterus of a third sheep
Surrogate mother
Embryonic development
RESULTS
Lamb (Dolly) genetically identical to mammary
cell donor
66
  • ? Since 1997, cloning has been demonstrated in
    many mammals, including mice, cats, cows, horses,
    mules, pigs, and dogs
  • ? CC (for Carbon Copy) was the first cat cloned
    however, CC differed somewhat from her female
    parent
  • ? Cloned animals do not always look or behave
    exactly the same

67
Figure 20.20
68
Problems Associated with Animal Cloning
  • ? In most nuclear transplantation studies, only a
    small percentage of cloned embryos have developed
    normally to birth, and many cloned animals
    exhibit defects
  • ? Many epigenetic changes, such as acetylation of
    histones or methylation of DNA, must be reversed
    in the nucleus from a donor animal in order for
    genes to be expressed or repressed appropriately
    for early stages of development

69
Stem Cells of Animals
  • ? A stem cell is a relatively unspecialized cell
    that can reproduce itself indefinitely and
    differentiate into specialized cells of one or
    more types
  • ? Stem cells isolated from early embryos at the
    blastocyst stage are called embryonic stem (ES)
    cells these are able to differentiate into all
    cell types
  • ? The adult body also has stem cells, which
    replace nonreproducing specialized cells

70
Figure 20.21
Embryonic stem cells
Adult stem cells
Cells generating all embryonic cell types
Cells generating some cell types
Cultured stem cells
Different culture conditions
Liver cells
Blood cells
Nerve cells
Different types of differentiated cells
71
  • ? Researchers can transform skin cells into ES
    cells by using viruses to introduce stem cell
    master regulatory genes
  • ? These transformed cells are called iPS cells
    (induced pluripotent cells)
  • ? These cells can be used to treat some diseases
    and to replace nonfunctional tissues

72
Figure 20.22
Remove skin cells from patient.
Reprogram skin cells so the cells become induced
pluripotent stem (iPS) cells.
Patient with damaged heart tissue or other disease
Treat iPS cells so that they differentiate into a
specific cell type.
Return cells to patient, where they can
repair damaged tissue.
73
Applications of DNA Technology
  • ? Medicine / Pharmaceutical
  • 1) Diagnosis of disease
  • 2) Human gene therapy
  • 3) Pharmaceutical products
  • -insulin, growth hormone, TPA (dissolves blood
    clots), proteins that mimic cell surface
    receptors for viruses like HIV

74
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75
Applications of DNA Technology
  • ? Forensic uses (PCR, DNA fingerprinting to match
    a suspect to DNA found at the scene of the crime)
  • ? Environmental uses microorganisms engineered
    to break down sewage, oil spills, etc.

76
O.J. Simpson capital murder case,1/95-9/95
  • ? Odds of blood in Ford Bronco not being R.
    Goldmans
  • ? 6.5 billion to 1
  • ? Odds of blood on socks in bedroom not being N.
    Brown-Simpsons
  • ? 8.5 billion to 1
  • ? Odds of blood on glove not being from R.
    Goldman, N. Brown-Simpson, and O.J. Simpson
  • ? 21.5 billion to 1
  • ? Number of people on planet earth
  • ? 6.1 billion
  • ? Odds of being struck by lightning in the U.S.
  • ? 2.8 million to 1
  • ? Odds of winning the Powerball lottery
  • ? 76 million to 1
  • ? Odds of getting killed driving to the gas
    station to buy a lottery ticket
  • ? 4.5 million to 1
  • ? Odds of seeing 3 albino deer at the same time
  • ? 85 million to 1
  • ? Odds of having quintuplets
  • ? 85 million to 1
  • ? Odds of being struck by a meteorite

77
Applications of DNA Technology
  • ? Agricultural uses
  • 1) livestock (bGH to enhance milk prod.)
  • 2) genetically engineered plants (resistant to
    herbicides pests, prevent spoilage, etc.)
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