Biotechnology and Genetic Engineering

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Biotechnology andGenetic Engineering

• AP Biology Chapter 20

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Terminology

• Genetic engineering direct manipulation of
  genetic material for practical purposes
• Biotechnology use of living organisms or their
  components to make products for us
• Recombinant DNA combining pieces of DNA from
  different organisms
• Gene cloning making copies of DNA

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Making recombinant DNA

• Plasmids (small circular pieces of DNA in
  bacterial cells) are used to insert pieces of
  foreign DNA

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The DNA is cut using restriction enzymes
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What are restriction enzymes?

• Restriction enzymes come from bacteria and
  recognize a particular pattern of DNA, often 4, 6
  or 8 base pairs long, and then cut the DNA within
  this recognized sequence.
• Bacteria use these enzymes to kill off other
  competing bacteria by cutting up their DNA.

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How do they cut?
BLUNT ENDS
STICKY ENDS
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• ACT GAA TTC CGG AAT GAA TTC
• TGA CTT AAG GCC TTA CTT AAG
• Where would the enzyme EcoRI cut?

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• ACT GAA TTC CGG AAT GAA TTC
• TGA CTT AAG GCC TTA CTT AAG

There would be three pieces one 4 bases, one
12 bases, and one 5 bases.
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How do bacteria protect its own DNA from being
cut by the enzymes?

• It methylates its own DNA.

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Making recombinant DNA in plasmids
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udent_view0/chapter18/steps_in_cloning_a_gene.html
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http//www.nearingzero.net/natural/screenres/natur
al039.jpg
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• Bacterial plasmids often contain
  antibiotic resistance genes.

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Genes can be cloned into vectors such as plasmids
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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
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Fig. 20-2a
Cell containing geneof interest
Bacterium
1
Gene inserted intoplasmid
Bacterialchromosome
Plasmid
Gene ofinterest
RecombinantDNA (plasmid)
DNA of chromosome
2
2
Plasmid put intobacterial cell
Recombinantbacterium
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Fig. 20-2b
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
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Fig. 20-4-1
Hummingbird cell
TECHNIQUE
Bacterial cell
lacZ gene
Restrictionsite
Gene of interest
Stickyends
Bacterial plasmid
ampR gene
Hummingbird DNA fragments
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Fig. 20-4-2
Hummingbird cell
TECHNIQUE
Bacterial cell
lacZ gene
Restrictionsite
Gene of interest
Stickyends
Bacterial plasmid
ampR gene
Hummingbird DNA fragments
Nonrecombinant plasmid
Recombinant plasmids
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Fig. 20-4-3
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
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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
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Steps

• Plasmid and DNA of gene of interest are isolated.
• Both DNAs are cut with the same restriction
  enzyme.
• new DNA is ligated into plasmid
• Recombinant plasmids are inserted into bacterial
  cells.
• Plate bacteria on agar. Bacteria will express
  new genes.

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Plasmid Maps
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Plasmid MapsSometimes called restriction maps
are graphical representation of plasmids, that
show the locations of major identifiable
landmarks on DNA like restriction enzyme sites,
genes of interest, plasmid length etc.
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• The collection of thousands of clones of
  bacteria containing recombinant plasmids is
  called a genomic library.

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• In molecular biology, plasmid (or restriction)
  maps are used as a reference to engineer
  plasmids.
• The plasmids are digested by enzymes chosen and
  the resulting samples are subsequently run on an
  electrophoresis gel.

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Our experiment to transform E.coli with pGLO
plasmid containing the jellyfish gene GFP to make
them have the ability to glow
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• To isolate only the cells containing the pGLO
  DNA, the plasmid contains the beta-lactamase gene
  which encodes for an ampicillin resistance (Ampr)
  protein.
• After the transformation, the cells are grown on
  a solid medium called an agar plate. This medium
  will contain the antibiotic ampicillin.
• In the presence of the ampicillin, only the
  bacteria containing the pGLO plasmid will have
  the Ampr protein which will break down the
  antibiotic, and be able to grow.
• This process is called antibiotic selection.

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GFP results in E.coli
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This plate shows bacteria expressing six
different types of flourescent proteins
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GRP has been used as tracers to see if the
plasmid has been taken up by the bacteria.
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How much does it cost to make a transgenic mouse?

• Transgenic Mouse Production The current fee for
  transgenic mouse production for UTMB
• investigators is 4000 (for 3 days of injections
  into C57BL/6 X C3H/HeF2 embryos) or 5200
• (for 4 days of injection into C57BL/6 or FVB/N
  embryos 1250 for each additional day

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Storing Cloned Genes in DNA Libraries

• Plasmid libraries containing genes of interest
  cloned in
• Phage library that is made using bacteriophages
  which store genes of interest

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Fig. 20-5a
Foreign genomecut up withrestrictionenzyme
or
Recombinantphage DNA
Bacterial clones
Recombinantplasmids
Phageclones
(a) Plasmid library
(b) Phage library
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Viruses used as vectors
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• BACs (bacterial artificial chromosome) are
  another type of vector used in DNA library
  construction
• A bacterial artificial chromosome (BAC) is a
  large plasmid that has been trimmed down and can
  carry a large DNA insert

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Fig. 20-5
Foreign genomecut up withrestrictionenzyme
Large insertwith many genes
Large plasmid
or
BACclone
Recombinantphage DNA
Bacterial clones
Recombinantplasmids
Phageclones
(a) Plasmid library
(b) Phage library
(c) A library of bacterial artificial
chromosome (BAC) clones
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• A complementary DNA (cDNA) library is made by
  cloning DNA made in vitro by reverse
  transcription of all the mRNA produced by a
  particular cell
• A cDNA library represents only part of the
  genomeonly the subset of genes transcribed into
  mRNA in the original cells

http//glencoe.mcgraw-hill.com/sites/9834092339/st
udent_view0/chapter18/cdna.html
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Fig. 20-6-1
DNA innucleus
mRNAs in cytoplasm
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Fig. 20-6-2
DNA innucleus
mRNAs in cytoplasm
Reversetranscriptase
Poly-A tail
mRNA
Primer
DNAstrand
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Fig. 20-6-3
DNA innucleus
mRNAs in cytoplasm
Reversetranscriptase
Poly-A tail
mRNA
Primer
DNAstrand
DegradedmRNA
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Fig. 20-6-4
DNA innucleus
mRNAs in cytoplasm
Reversetranscriptase
Poly-A tail
mRNA
Primer
DNAstrand
DegradedmRNA
DNA polymerase
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Fig. 20-6-5
DNA innucleus
mRNAs in cytoplasm
Reversetranscriptase
Poly-A tail
mRNA
Primer
DNAstrand
DegradedmRNA
http//glencoe.mcgraw-hill.com/sites/9834092339/st
udent_view0/chapter18/fish.html
DNA polymerase
cDNA
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Ways to introduce new genes into bacteria.
Conjugation through tubes between bacteria
Transformation negative DNA taken
up Transduction by bacteriophages or other
viruses Mutation ALL of these introduce GENETIC
VARIATION!
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Nucleic Acid Hybridization

• Used to detect genes
• The DNA of the cell is denatured to produce
  single stranded DNA.
• The radioactive probe will hybridize (bond) with
  complementary bases if present.
• Probes can be radioactive isotopes or
• flourescent dyes.

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The radioactive probe is made by determining a
short segment of the protein sequence, then "back
translating" to the possible short DNA sequences
called oligomers. Then these DNA oligomers
(known as "oligos") are radiolabeled, and applied
to the blotted clones.  They should hybridize
only to clones containing sequence encoding the
desired protein.
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How does this work?

• Protein segment
• gly gly ser glutamic acid
• Look on Genetic Codon chart, find mRNA codons
• GGU GGU UCU GAA
• Make a radioactive DNA
• (oligos)probe
• CCA CCA AGA - CTT

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Expression of eukaryoticgenes in prokaryotes

• Use an expression vector with a prokaryotic
  promoter upstream from the location of the gene
  (ie operon)
• Create artificial genes without introns since
  bacteria do not have the machinery for
  eliminating introns.
• YACS

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What are YACS?

• Yeast artificial chromosomes that carry foreign
  DNA.
• Yeast cells have plasmids that can act as vectors.

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Electroporation

• injecting DNA into eukaryotic cells

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PCR Polymerase Chain Reaction

• Used to amplify DNA
• Discovered by Kary Mullis (GT grad)

A Thermocycler
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Steps of PCR?

• Denature DNA (94-96 C)
• Anneal (base pair) primers (50 65 C)
• Extend primers (72 for polymerase to work)
• Machines called thermocyclers do this.

http//glencoe.mcgraw-hill.com/sites/9834092339/st
udent_view0/chapter18/polymerase_chain_reaction.ht
ml
http//www.dnalc.org/ddnalc/resources/shockwave/pc
ranwhole.html
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Fig. 20-8b
5?
3?
1
Denaturation
3?
5?
Annealing
2
Cycle 1yields 2 molecules
Primers
3
Extension
Newnucleo-tides
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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
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• In PCR, a heat-stable DNA polymerase is used,
  most commonly Taq Polymerase from the
  thermophilic microbe Thermus aquaticus. 
• Thomas Brock discovered T. aquaticus  from a hot
  spring at Yellowstone National Park.

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Applications of PCR

• PCR has replaced cloning for many purposes,
  particularly the sequencing of DNA. 
• It is faster and requires no vectors, which can
  mutate as they reproduce.  
• It can be used forensically, to amplify tiny
  amounts of DNA from criminal evidence or
  clinically, to detect DNA sequences linked to
  inherited disorders.  

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What is gel electrophoresis?

• A technique to separate DNA based on the movement
  of DNA fragments from neg to pos (DNA is neg).
• Smaller fragments travel farther.
• Samples are placed in gels.

Gel Electrophoresis
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Southern BlottingDNA Fingerprinting- named
for Edwin Southern- used to identify DNA
fragments

• Isolate DNA
• Cut DNA into fragments with restriction enzymes.
• Electrophorese.
• Blot onto nylon membrane.
• Apply radioactive probes.
• Wash to remove unbonded probes.

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tudent_view0/chapter14/animation_quiz_5.html
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In DNA fingerprinting

• Single nucleotide polymorphisms (SNPs) are useful
  genetic markers
• These are single base-pair sites that vary in a
  population (most of our DNA is identical

http//glencoe.mcgraw-hill.com/sites/9834092339/st
udent_view0/chapter18/restriction_fragment_length_
polymorphisms.html
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Many genetic diseases are the result of a
polymorphism at a single locus.
Fig. 20-21
DNA
T
Normal allele
SNP
C
Disease-causingallele
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udent_view0/chapter18/video_quiz_-_world_trade_cen
ter_dna.html
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• When a restriction enzyme is added, SNPs result
  in DNA fragments with different lengths, or
  restriction fragment length polymorphisms (RFLP)
• Some polymorphisms cause disease, while some do
  not. Others indicate a predisposition to
  disease.

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Hemoglobin S is missing a restriction site due to
a polymorphism
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RFLP Analysis in Paternity Cases
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RFLP Analysis in Paternity Cases
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Short Tandem Repeats

• Even more sensitive is the use of genetic markers
  called short tandem repeats (STRs), which are
  variations in the number of repeats of specific
  DNA sequences

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• The Human Genome Project has shown that there are
  tens of thousands of STR loci in human DNA.
• An individual inherits one copy of an STR from
  each parent,

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D7S280 is one of the 13 core CODIS STR genetic
loci. This DNA is found on human chromosome 7.
The DNA sequence of a representative allele of
this locus is shown below. The tetrameric repeat
sequence of D7S280 is "gata". Different alleles
of this locus have from 6 to 15 tandem repeats of
the "gata" sequence. How many tetrameric repeats
are present in the DNA sequence shown below?
1 aatttttgta ttttttttag agacggggtt tcaccatgtt
ggtcaggctg actatggagt 61 tattttaagg ttaatatata
taaagggtat gatagaacac ttgtcatagt ttagaacgaa121
ctaacgatag atagatagat agatagatag atagatagat
agatagatag atagacagat181 tgatagtttt tttttatctc
actaaatagt ctatagtaaa catttaatta ccaatatttg241
gtgcaattct gtcaatgagg ataaatgtgg aatcgttata
attcttaaga atatatattc301 cctctgagtt tttgatacct
cagattttaa ggcc


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If the genotypes of both parents are known, we
use a Punnett Square to predict the possible
phenotypes of their offspring. Each child
inherits one allele of a given locus from each
parent. Panel (a) - At the D21S11 locus, the
children of Bob Blackett and wife Anne can have
four different genotypes. Son David is 28, 31.
Daughter Katie is 29, 30. Panel (b) - Bob
Blackett inherited the 31 allele from his mother,
Norma. Therefore the 29 allele is paternal. If
Bob's paternal was not 29, what would be your
conclusion?
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Huntingtons Disease can be diagnosed by the
number of CAG repeats
The data below shows the results of
electrophoresis of PCR fragments amplified using
probes for the site which has been shown to be
altered in Huntington's disease. The male parent,
as shown by the black box, got Huntington's
disease when he was 40 years old. His children
include 6 (3,5,7,8,10,11) with Huntington's
disease, and the age at which the symptoms first
began is shown by the number above the band from
the PCR fragment.
What is the prognosis for the normal children 4,
6, and 9?
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Sanger Sequencing

• Used to sequence short segments of DNA
• Single-stranded fragments are incubated with
  fluorescent-tagged short segments for DNA
  hybridization.
• When fragments hybridize with the tagged
  nucleotide, the hybridization stops.
• Fragments are electrophoresed and analyzed.

http//glencoe.mcgraw-hill.com/sites/9834092339/st
udent_view0/chapter18/sanger_sequencing.html
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Analyzing Expression of Genes

• Northern Blotting using radioactive probes to
  look for mRNA being produced
• RT-PCR Reverse transcriptase-polylmerase chain
  reaction makes cDNA from mRNAs and then PCRs
  the DNA for electrophoresis
• in situ hybridization can locate specific
  mRNAs in cells

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• Micro arrays - Isolate mRNA from cells, make
  cDNA using reverse transcriptase, then uses cDNA
  to explore collections of genomic DNA to see if
  they hybridize

http//glencoe.mcgraw-hill.com/sites/9834092339/st
udent_view0/chapter18/using_a_dna_microarray.html
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• Microarrays are useful in discerning gene
  expression in different tissues AND at different
  stages of development.
• Different brightness and
• colors signify rates of
• expression.

http//www.dnalc.org/resources/3d/26-microarray.ht
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Google Image Result for http//www.g2conline.info/
content/1178/1178_what_microarray_thumb.jpg
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An example
DNA Microarray Methodology Animation
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Determining Gene Function

• In vitro mutagenesis changes made to cloned
  gene, gene returned to cell and it knocks out
  the normal gene. Then look for abnormalities.
• RNA interference (RNAi) uses RNA to block
  translation of mRNA and see what happens.

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Cloning Organisms

• Organismal cloning producing genetically
  identical individuals from a single somatic cell
  of a multicellular organism

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In plants

• Steward demonstrated genomic equivalence in
  plants by growing carrot plants from
  differentiated root cells.
• Most plant cells remain totipotent, retaining the
  ability to give rise to a complete new organisms.

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In Animals

• Briggs and all transplanted nuclei from embryonic
  frog cells into enucleated egg cells and produced
  cloned frogs
• Nuclear transplantation name of process
• Whether normal development occurred depended on
  developmental age of the transplanted nucleus.

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Fig. 20-17
Frog embryo
Frog egg cell
Frog tadpole
EXPERIMENT
UV
Fully differ- entiated (intestinal) cell
Less differ-entiated cell
Donornucleustrans-planted
Donor nucleus trans- planted
Enucleated egg cell
Egg with donor nucleus activated to begin
development
RESULTS
Most develop into tadpoles
Most stop developing before tadpole stage
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Nuclear Transplantation
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And then Dolly came along in 1997
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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
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Why Dolly died young 6 yrs

• Dolly's telomeres were found to be approximately
  80 of the length they should be for a sheep her
  age.
• Also there is the concern of damaged DNA being
  carried into the clone

95

• Cloned animals do not look exactly like the
  transplanted nucleus due to cytoplasmic affects.

CC
Rainbow
Hi Mrs. Smith!
CC and her Surrogate mom
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• In most nuclear transplantation studies, only a
  small percentage of cloned embryos have developed
  normally to birth
• 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

• Relatively unspecialized cells that continue to
  reproduce themselves and can be induced to form
  specialized cells
• Embryonic cells are more totipotent than adult
  stem cells

http//cbm.msoe.edu/stupro/so/SOStemCellVideo2010.
html
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• Therapeutic cloning using stem cells to replace
  organs and tissues
• Reproductive cloning using stem cells to
  reproduce new organisms
• Both raise ethical
• debates

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Research points to a new direction in Stem Cell
Research
Induced Pluripotent Stem Cells
102
The Nobel Prize in Medicine 2012 was awarded
to two biologists for their breakthroughs in the
field of stem-cell research two discoveries
that happened 44 years apart. The honors go to
Britain's Sir John B. Gurdon and Japan's Shinya
Yamanaka for their pioneering work with the
life-shaping cells, which can be reprogrammed to
create any kind of tissue in the body.
103
Benefits of DNA technology

• Medical Applications
• identification of human genes in which mutation
  plays a role in genetic diseases

104
Huntingtons Disease can be diagnosed by the
number of CAG repeats
The data below shows the results of
electrophoresis of PCR fragments amplified using
probes for the site which has been shown to be
altered in Huntington's disease. The male parent,
as shown by the black box, got Huntington's
disease when he was 40 years old. His children
include 6 (3,5,7,8,10,11) with Huntington's
disease, and the age at which the symptoms first
began is shown by the number above the band from
the PCR fragment.
What is the prognosis for the normal children 4,
6, and 9?
105
Human Gene Therapy

• Gene therapy is the alteration of an afflicted
  individuals genes
• Vectors, such as viruses, are used for delivery
  of genes into specific types of cells, for
  example bone marrow
• It may be difficult to target cells.
• Gene therapy raises ethical questions, such as
  whether human germ-line cells should be treated
  to correct the defect in future generations

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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
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Pharmaceutical Products

• Advances in DNA technology and genetic research
  are important to the development of new drugs to
  treat diseases
• In particular pharm animals and plants can be
  used to produce certain products

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Fig. 20-23
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Forensic Evidence and Genetic Profiles

• An individuals unique DNA sequence, or genetic
  profile, can be obtained by analysis of tissue or
  body fluids

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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.
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Environmental Cleanup

• Some modified microorganisms can be used to
  extract minerals from the environment or degrade
  potentially toxic waste materials
• Biofuels make use of crops such as corn,
  soybeans, and cassava to replace fossil fuels

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Genetic Engineering in Plants

• Agricultural scientists have endowed a number of
  crop plants with genes for desirable traits
• The Ti plasmid is the most commonly used vector
  for introducing new genes into plant cells

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Is this safe?

• Most public concern about possible hazards
  centers on genetically modified (GM) organisms
  used as food
• Some are concerned about the creation of super
  weeds from the transfer of genes from GM crops
  to their wild relatives

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Fig. 20-25
TECHNIQUE
Agrobacterium tumefaciens
Tiplasmid
Site whererestrictionenzyme cuts
T DNA
RESULTS
DNA withthe geneof interest
RecombinantTi plasmid
Plant with new trait
115

• Guidelines are in place in the United States and
  other countries to ensure safe practices for
  recombinant DNA technology