Chapter 14
Biotechnology and Genomics
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Mills AP Bio 2012-2013
Chapter 14 Biotechnology and Genomics
• Topics
– 14.1
– 14.2
– 14.3
– 14.4
Cloning of a gene
Biotechnology products
Gene therapy
Genomics
Mills AP Bio 2012-2013
Chapter 14 Biotechnology and Genomics
14.1 Cloning of a Gene
• Cloning of a Gene
– Two basic ways to clone a gene:
• Recombinant DNA –
– when need very large quantities.
• Polymerase Chain Reaction (PCR)
– When need lesser quantities
– Done in a test tube
– Genomic Library
– Analyzing DNA
Mills AP Bio 2012-2013
Chapter 14 Biotechnology and Genomics
14.1 Cloning of a Gene
• Recombinant DNA
– DNA from two different sources is
combined.
– Need a vector to get the new
DNA into the organism
• Often use plasmids or bacteriophage
– Need enzymes to get the new
DNA into the vector.
• Restriction enzyme cuts the DNA of
the plasmid.
• DNA ligase seals the new DNA into
the plasmid.
Mills AP Bio 2012-2013
Mills AP Bio 2012-2013
Chapter 14 Biotechnology and Genomics
14.1 Cloning of a Gene
• Restriction enzymes
– May occur naturally.
– Bacteria use as a defense against
foreign viruses.
– Hamilton Smith isolated the first
restriction enzyme in 1970, since
then hundreds have been isolated.
Hamilton Smith 19321978 Nobel Prize in
Physiology or Medicine
Mills AP Bio 2012-2013
Chapter 14 Biotechnology and Genomics
14.1 Cloning of a Gene
• EcoR1
– Example of a restriction enzyme.
– “sticky ends” will bind to “sticky ends” of new DNA, with the
help of DNA ligase to form rDNA.
– Always cuts between the A and the G.
– Recognizes the palindrome GAATTC (CTTAAG).
Mills AP Bio 2012-2013
Mills AP Bio 2012-2013
Mills AP Bio 2012-2013
Chapter 14 Biotechnology and Genomics
14.1 Cloning of a Gene
• Cloning of a Gene
– Using restriction
enzymes (cont)
Mills AP Bio 2012-2013
Chapter 14 Biotechnology and Genomics
14.1 Cloning of a Gene
• Plasmid Vector
Compared to Viral
(bacteriophage) Vector
• Bacteria cannot deal
with introns – what
problems does that
present?
• How is that fixed?
Mills AP Bio 2012-2013
Mills AP Bio 2012-2013
Chapter 14 Biotechnology and Genomics
14.1 Cloning of a Gene
• PCR
– Can create millions of copies of
a single gene or sequence very
quickly.(PCR usually used to
create more “genes”, gene
cloning usually used to make
more “gene product”)
– Still use rDNA cloning if need
large quantities of gene product.
– Very specific.
– Done in a test tube.
– Needs primers and DNA
polymerase.
– Used to have to keep adding
more enzyme each time the
DNA was heated to separate
strands – found a heat
insensitive enzyme.
Mills AP Bio 2012-2013
PCR
PCR tutorial:..\Biology Clipart Movies Animations Sounds\Biology tutorials\pcr .dir
Mills AP Bio 2012-2013
Mills AP Bio 2012-2013
Chapter 14 Biotechnology and Genomics
14.1 Cloning of a Gene
• Analyzing DNA- Gel Electrophoresis
– Entire genome of an individual organism
can be categorized.
– Each individual will have a unique “DNA
fingerprint”.
– Whole genome treated with restriction
enzymes. End up with many different
length DNA fragments
• Different individuals have some
fragments of DNA that are different
lengths than another individuals =
restriction fragment length polymorphisms
(RFLP’s).
– Fragments separated on basis of length
(weight).
– More commonly, use only certain
fragments (short tandem repeats –
STRs) of interest that have been
amplified by PCR.
Mills AP Bio 2012-2013
Electrophoresis tutorial on HD.\AP Bio
Lab\electrophoresis_tutorial.swf
http://learn.genetics.utah.edu/content/labs/gel/
DNA gel
Mills AP Bio 2012-2013
Mills AP Bio 2012-2013
Chapter 14 Biotechnology and Genomics
14.1 Cloning of a Gene
•
Quagga related to zebra not horse
– Became extinct in 1870
– Used DNA from dried skin
•
DNA from Sam Sheppard
– forensics
•
Human mummies
– Determine evolutionary history of human populations
Mills AP Bio 2012-2013
Chapter 14 Biotechnology and Genomics
14.1 Cloning of a Gene
Lane 1 and Lane 4 -
– Results from a single
probe
Because locus
these lanes
have
bands in common,
DNA fingerprint no
analysis
for a man
neither lane can contain the
and his four different
children are
DNA of the father. Lane 3 is
shown in the figure.
Which lane
the father.
contains the DNA of the father?
– Each parent contributes
Lane 2 andone
Lane set
5 - of
Because
these
lanes have
alleles to each child.
The
autorad
no bands in common,
above contains DNA
from
four
neither
lane can
contain the
of theEach
father. child
children and one DNA
father.
will share one band (allele) with the
father. You can use the process of
elimination to determine which lane
contains the father's DNA.
Mills AP Bio 2012-2013
Chapter 14 Biotechnology and Genomics
14.2 Biotechnology Products
• Biotechnology
Products
–
–
–
–
Genetically Modified Bacteria
Genetically Modified Plants
Genetically Modified Animals
Cloning of Transgenic
Animals
Mills AP Bio 2012-2013
Organisms that have
had foreign DNA
inserted are called
transgenic organisms
Chapter 14 Biotechnology and Genomics
14.2 Biotechnology Products
• Transgenic Bacteria
– Genetically altered
bacteria are used to
produce many things,
such as:
• Insulin
• Human growth factor
• Tissue plasminogen
activator
• Hepatitis vaccine
– Genetically altered
bacteria is a big industry.
• Some examples of things
transgenic bacteria can
do:
– Eat oil from oil spills
– Protect plants from
insects
– Remove sulfur from coal
– Act as biofilters for air
– Produce phenylalanine
(used in NutriSweet)
Mills AP Bio 2012-2013
Chapter 14 Biotechnology and Genomics
14.2 Biotechnology Products
Mills AP Bio 2012-2013
Chapter 14 Biotechnology and Genomics
14.2 Biotechnology Products
• Transgenic Plants
– Many crops have been altered to be
insect and or herbicide resistant
• Cotton, corn, potato, working on wheat
and rice
– Can increase protein or starch content
among other things.
– Plants can be made to produce some
hormones, clotting factors and
antibodies.
– Little chance of contamination with
pathogens that could infect humans.
Mills AP Bio 2012-2013
Flavr Savr Tomato – first
genetically altered food
approved by FDA in
1994.
Chapter 14 Biotechnology and Genomics
14.2 Biotechnology Products
Flavr Savr
Traditional
The
traditional
tomato must
be
harvested
while it is
still green
and firm so
that it is not
crushed on
the way to
the
supermarkt.
The Flavr
Savr tomato
ripens on
the vine –
resulting in
fuller
flavour. It is
modified so
that it
remains firm
after
harvesting.
Supermarket
Ripe and
Increased
Flavour.
Mills AP Bio 2012-2013
Chapter 14 Biotechnology and Genomics
14.2 Biotechnology Products
• Transgenic Animals
– Insert desired gene into
animal’s egg – fertilize in vitro
– implant – see gene’s
effects in the new born
animal.
– Can grow bigger animals by
inserting gene for bovine
growth hormone.
– Gene “pharming”
• Use animals to “grow”
pharmaceuticals
• Goats, mice, cows
Mills AP Bio 2012-2013
Herd of goats exists that
produces Antithrombin
II(anticlotting agent) in
their milk.
Chapter 14 Biotechnology and Genomics
14.2 Biotechnology Products
Mills AP Bio 2012-2013
Chapter 14 Biotechnology and Genomics
14.2 Biotechnology Products
Mills AP Bio 2012-2013
Chapter 14 Biotechnology and Genomics
14.2 Biotechnology Products
• Cloning
Transgenic
Animals
– Best way to
get identical
animal.
Sexual
reproduction
would lead to
variety.
Mills AP Bio 2012-2013
Chapter 14 Biotechnology and Genomics
14.3 Gene Therapy
• Gene Therapy
– Genetic Disorders
• Cystic fibrosis
• SCID (severe combined
immunodificiency
syndrome)
• Familial
hypercholesterolemia
– Cancer treatment
• Make normal cells more
tolerant of the
chemotherapy
– Other Illnesses
– Limited success – so far.
Mills AP Bio 2012-2013
Mills AP Bio 2012-2013
Chapter 14 Biotechnology and Genomics
14.3 Gene Therapy
• Cancer
– Make bone marrow less
susceptible to the
effects of
chemotherapy?
– Infect with normal p53
gene?
– Who knows what the
future holds?
Mills AP Bio 2012-2013
Chapter 14 Biotechnology and Genomics
14.4 Genomics
• Gene therapy involves procedures to give
patients healthy genes to make up for a faulty
gene.
• It also includes the use of genes to treat
genetic disorders and various human
illnesses.
• There are ex vivo (outside body) and in vivo
(inside body) methods of gene therapy.
– Ex Vivo
• Children with Severe Combined Immunodeficiency
(SCID) lack the enzyme adenosine deaminase
(ADA)
• Bone marrow stem cells are infected with a virus
carrying a normal gene for the ADA enzyme
– In Vivo
• Cystic Fibrosis
• Nasal / Respiratory Spray
Mills AP Bio 2012-2013
Chapter 14 Biotechnology and Genomics
14.4 Genomics
• Human Genome Project
– Launched in 1990 by NIH and
DOE (Dept of Energy)
– Two goals
• Construct a genetic map of the
human genome
• Construct a base sequence
map (completed in 2001)
• Took 13 years to sequence
three billion base pairs along
the length of chromosomes.
– Have sequences for fruit fly,
bakers yeast, a roundworm and
about 30 bacteria –and now
Human.
– New field called Genomics.
Mills AP Bio 2012-2013
•
http://en.wikipedia.org/wiki/Human_genome
– Humans have about 20,500 genes (accounting for only 1.5-2% of all DNA)
• Most code for proteins
• 95% of the average protein-coding gene in humans is introns
• Much of the human genome was formerly described as “junk”
– Does not specify the order of amino acids in a polypeptide
AP Bioeffect
2012-2013
– RNA molecules can haveMills
a regulatory
in cells
– 3 billion bases – would take 200 books each 1000 pages long to hold
• Eukaryotic Gene Structure
– Historically, genes were defined as
discrete units of heredity that corresponded
to a locus on a chromosome.
– Prokaryotes typically possess a single
circular chromosome.
– Eukaryotic chromosomes are much more
complex.
• Genes are distributed along the length of a
chromosome.
• Genes are fragmented into exons.
36
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
exon
exon
repetitive and unique DNA
intron
Human Genome
Gene B
Gene A
exon
exon
pre- mRNA
pre-mRNA
intro n
removed
re mo ve d
RNA
introns
5′
3′
Gene A mRNA
5′
3′
Gene B mRNA
37
• Eukaryotic Gene Structure
– Intergenic sequences are
DNA sequences that occur
between genes
– Repetitive DNA elements
occur when the same
sequence of two or more
nucleotides is repeated many
times along the length of one
or more chromosomes.
– Transposons are specific
DNA sequences that have the
remarkable ability to move
within and between
chromosomes.
38
Mills AP Bio 2012-2013
• Functional Genomics
– Functional genomics aims to understand the role of the genome in
cells or organisms
– DNA microarrays can monitor the expression of thousands of genes
simultaneously and tell us:
• What genes are turned on
• Environmental conditions that turn on the gene
40
• Functional Genomics
– DNA microarrays contain microscopic amounts of
known DNA fixed onto a small glass slide or
silicon chip in known locations
– mRNAs bind to DNA sequences on the chip
through complementary base pairing
• Allows identification of genes that are active in the cell
– Can be used to identify various mutations in the
genome of an individual
• This is called the person’s genetic profile.
41
DNA Microarray Technology
tagged DNA did
bind to probe
DNA probe
tagged
DNA
tagged DNA did not
bind to probe
testing subject's DNA
© Deco/Alamy
42
• Comparative genomics compares the human genome to the
genomes of other organisms
• Model organisms have genetic mechanisms and cellular
pathways in common with humans
• Comparative genomics offers a way to study changes in the
genome over time
45
• Proteomics - The study of the structure,
function, and interaction of cellular proteins
• The entire collection of a species’ proteins is
its proteome
• The proteome is larger than the genome
– Mechanisms such as alternative -re-mRNA splicing
increase the number of possible proteins
• Understanding protein function is essential to
the development of better drugs
– Correlate drug treatment to the particular genome
– Increase efficiency and decrease side effects
• Once the primary structure of a protein is
known
– It should be possible to predict its tertiary structure
– Computer modeling of the tertiary structures of
proteins is an important part of proteomics
46
• Bioinformatics - the application of computer
technologies, software, and statistical techniques to
the study of biological information
– Genomics and proteomics produce raw data
– These fields depend on computer analysis to find significant
patterns in the data
– Scientists hope to find relationships between genetic profiles and
genetic disorders
• New computational tools will be needed to accomplish
these goals
47
Chapter 14 Biotechnology and Genomics
14.4 Genomics
• How to sequence DNA (if time)
– http://www.dnalc.org/ddnalc/resources/cyc
seq.html
Mills AP Bio 2012-2013
The End
Mills AP Bio 2012-2013