NOTES - CH 20: DNA Technology

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NOTES - CH 20 DNA Technology
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• ? 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

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

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Recombinant DNA

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

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

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• 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

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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

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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

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• 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

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• 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.

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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
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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?

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Bacterial plasmids in gene cloning
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DNA Analysis Genomics

• ? PCR (polymerase chain reaction)
• ? Gel electrophoresis
• ? Restriction fragment analysis (RFLPs)
• ? Southern blotting
• ? DNA sequencing
• ? Human genome project

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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
•  

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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!!
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Polymerase Chain Reaction (PCR)

• ? PCR is highly specific primers determine the
  sequence to be amplified
• ? only tiny amounts of DNA are needed

Remember these?
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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!)

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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!!!

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5?
3?
TECHNIQUE
Targetsequence
Genomic DNA
5?
3?
Denaturation
5?
3?
5?
3?
Annealing
Cycle 1yields2molecules
Primers
Extension
Newnucleotides
Cycle 2yields4molecules
Cycle 3yields 8molecules2 molecules(in white
boxes)match targetsequence
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5?
3?
TECHNIQUE
Targetsequence
Genomic DNA
5?
3?
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Figure 20.8b
5?
3?
Denaturation
3?
5?
Annealing
Cycle 1yields2molecules
Primers
Extension
Newnucleo-tides
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Figure 20.8c
Cycle 2yields4molecules
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Figure 20.8d
Cycle 3yields 8molecules2 molecules(in white
boxes)match targetsequence
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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.

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PCR works like a copying machine for DNA!
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DNA Analysis

• ? Gel electrophoresis separates nucleic acids
  or proteins on the basis of size or electrical
  charge creating DNA bands of the same length

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Restriction fragment analysis

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

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DNA Sequencing

• ? Determination of nucleotide sequences (Sanger
  method, sequencing machine)
• ? Human Genome Project

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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

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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
DNApolymerase
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 (templatestrand)
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
Directionof movementof strands
Longest labeled strand
Detector
Laser
Shortest labeled strand
RESULTS
Last nucleotideof longestlabeled strand
G
A
C
T
G
A
Last nucleotideof shortestlabeled strand
A
G
C
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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
DNApolymerase
dTTP
C
ddTTP
T
dGTP
ddGTP
T
C
G
P
P
P
P
P
P
A
G
G
C
A
OH
H
A
3?
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Figure 20.12b
TECHNIQUE (continued)
DNA (templatestrand)
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
Directionof movementof strands
Longest labeled strand
Detector
Laser
Shortest labeled strand
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Figure 20.12c
Directionof movementof strands
Longest labeled strand
Detector
Laser
Shortest labeled strand
RESULTS
Last nucleotideof longestlabeled strand
G
A
C
T
G
A
Last nucleotideof shortestlabeled strand
A
G
C
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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

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

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Figure 20.14
50 ?m
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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

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Figure 20.15
TECHNIQUE
Isolate mRNA.
Tissue sample
Make cDNA by reversetranscription,
usingfluorescently labelednucleotides.
mRNA molecules
Labeled cDNA molecules(single strands)
Apply the cDNA mixture to a microarray, a
different genein each spot. The cDNA
hybridizeswith any complementary DNA onthe
microarray.
DNA fragmentsrepresenting aspecific gene
DNA microarray
Rinse off excess cDNA scan microarrayfor
fluorescence. Each fluorescent spot(yellow)
represents a gene expressedin the tissue sample.
DNA microarraywith 2,400human genes
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Figure 20.15a
DNA microarraywith 2,400human genes
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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

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• ? 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

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• ? 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

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Figure 20.16
DNA
T
Normal allele
SNP
C
Disease-causingallele
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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

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Cloning Plants Single-Cell Cultures

• ? A totipotent cell is one that can generate a
  complete new organism
• ? Plant cloning is used extensively in agriculture

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Figure 20.17
Crosssection ofcarrot root
2-mgfragments
Single cellsfree insuspensionbegan todivide.
Embryonicplant developedfrom a culturedsingle
cell.
Fragments werecultured in nu-trient
mediumstirring causedsingle cells toshear off
intothe liquid.
Plantlet wascultured onagar medium.Later it
wasplanted in soil.
Adultplant
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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

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Figure 20.18
EXPERIMENT
Frog embryo
Frog egg cell
Frog tadpole
UV
Fully differ-entiated(intestinal) cell
Less differ-entiated cell
Donornucleustrans-planted
Donornucleustrans-planted
Enucleatedegg cell
Egg with donor nucleusactivated to
begindevelopment
RESULTS
Most stop developingbefore tadpole stage.
Most developinto tadpoles.
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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

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Figure 20.19
TECHNIQUE
Mammarycell donor
Egg cell donor
Eggcell fromovary
Nucleusremoved
Cells fused
Culturedmammarycells
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment
Lamb (Dolly) geneticallyidentical to mammary
cell donor
RESULTS
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Figure 20.19a
TECHNIQUE
Mammarycell donor
Egg cell donor
Eggcell fromovary
Nucleusremoved
Cells fused
Culturedmammarycells
Nucleus frommammary cell
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Figure 20.19b
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment
RESULTS
Lamb (Dolly) geneticallyidentical to mammary
cell donor
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• ? 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

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Figure 20.20
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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

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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

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Figure 20.21
Embryonicstem cells
Adultstem cells
Cells generatingall embryoniccell types
Cells generatingsome cell types
Culturedstem cells
Differentcultureconditions
Livercells
Bloodcells
Nervecells
Differenttypes ofdifferentiatedcells
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• ? 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

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Figure 20.22
Remove skin cellsfrom patient.
Reprogram skin cellsso the cells becomeinduced
pluripotentstem (iPS) cells.
Patient withdamaged hearttissue or otherdisease
Treat iPS cells sothat they differentiateinto a
specificcell type.
Return cells topatient, wherethey can
repairdamaged tissue.
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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

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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.

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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

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Applications of DNA Technology

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