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Chapter 17 – 18
Biotechnology:
Genomics & DNA Technology
DNA Sequencing
 Sanger method
determine the base sequence
of DNA
 dideoxynucleotides

 ddATP, ddGTP, ddTTP, ddCTP
 missing O for bonding of next
nucleotide
 terminates chain
DNA Sequencing
 Sanger method
synthesize
complementary DNA
strand in vitro
 in each tube:
1

 “normal” N-bases
 dideoxy N-bases
 ddA, ddC, ddG, ddT
 DNA polymerase
 primer
 buffers & salt
2
3
4
2
Fred Sanger
This was his 2nd Nobel Prize!!

1st was in 1958 for the
structure of insulin
1978 | 1980
Advancements to Sequencing
 Fluorescent tagging
no more radioactivity
 all 4 bases in 1 lane

 each base a different color
 Automated reading
Advancements to Sequencing
 Fluorescent tagging sequence data
 Computer read & analyzed
Raw Genome Data
Automated Sequencing Machines
 Really BIG labs!
Human Genome Project
 U.S government project

begun in 1990
 estimated to be a 15 year project

DOE & NIH
 initiated by Jim Watson
 led by Francis Collins

goal was to sequence entire
human genome
 3 billion base pairs
 Celera Genomics



Craig Venter challenged gov’t
would do it faster, cheaper
private company
Different Approaches
gov’t method
Craig Venter’s method
“map-based method”
“shotgun method”
1. Cut chromosomal DNA segment into
fragments, arrange based on
overlapping nucleotide sequences,
and clone fragments.
2. Cut and clone into smaller fragments.
3. Assemble DNA sequence
using overlapping sequences.
1. Cut DNA from entire chromosome
into small fragments and clone.
2. Sequence each segment & arrange
based on overlapping nucleotide
sequences.
Human Genome Project
On June 26, 2001, HGP published the “working
draft” of the DNA sequence of the human genome.
Historic Event!
blueprint
of a human
 the potential to
change science
& medicine

How does our genome stack up?
Organism
Human (Homo sapiens)
Laboratory mouse (M. musculus)
Mustard weed (A. thaliana)
Roundworm (C. elegans)
Fruit fly (D. melanogaster)
Yeast (S. cerevisiae)
Bacterium (E. coli)
Human Immunodeficiency Virus (HIV)
Genome Size
(bases)
Estimated
Genes
3 billion
30,000
2.6 billion
30,000
100 million
25,000
97 million
19,000
137 million
13,000
12.1 million
6,000
4.6 million
3,200
9700
9
GenBank
Database of
genetic
sequences
gathered
from
research
Publicly
available!
Organizing the Data
And we didn’t stop there…
Interspersed Repetitive DNA
 Repetitive DNA is spread throughout
genome
interspersed repetitive DNA (SINEs
Short INterspersed Elements) make up
25-40% of mammalian genome
 in humans, at least 5% of genome is
made of a family of similar sequences
called, Alu elements (PV92 anyone?!)

 300 bases long
 Alu is an example of a "jumping gene"
called a transposon; a DNA sequence that
"reproduces" by copying itself & inserting
into new chromosome locations
Rearrangements in the Genome
 Transposons

transposable genetic element
 piece of DNA that can move from one
location to another in cell’s genome
One gene of an insertion sequence codes for transposase, which catalyzes the
transposon’s movement. The inverted repeats, about 20 to 40 nucleotide pairs long,
are backward, upside-down versions of each other. In transposition, transposase
molecules bind to the inverted repeats & catalyze the cutting & resealing of DNA
required for insertion of the transposon at a target site.
Transposons
Insertion of
transposon
sequence in new
position in genome
Insertion sequences
cause mutations
when they happen to
land within the
coding sequence of a
gene or within a DNA
region that regulates
gene expression.
Transposons
 Barbara McClintock

1947 | 1983
discovered 1st transposons in Zea mays
(corn) in 1947
Retrotransposons
 Transposons actually make up over 50%
of the corn (maize) genome & 10% of the
human genome.
Most of these
transposons are
retrotransposons,
transposable
elements that move
within a genome by
means of RNA
intermediate,
transcript of the
retrotransposon
DNA.
Families of Genes
 Human globin gene family

evolved from duplication of common
ancestral globin gene
Different versions are
expressed at different
times in development
allowing hemoglobin to
function throughout life
of developing animal
Hemoglobin
Differential
expression of
different beta
globin genes
ensures
important
physiological
changes
during human
development.
The BIG Questions…
 How can we use our knowledge of DNA to:
diagnose disease or genetic defect?
 cure disease or genetic defect?
 change/improve organisms?

 What are the techniques & applications of
biotechnology?

direct manipulation of genes for practical
purposes
Biotechnology
 Genetic manipulation of organisms is
not new

humans have been doing this for
thousands of years
 plant & animal breeding
Evolution & Breeding of Food Plants
artificial selection!
Evolution of Zea mays from ancestral teosinte (left) to
modern corn (right). The middle figure shows possible
hybrids of teosinte & early corn varieties
Evolution & Breeding of Food Plants
 “Descendants” of the wild mustard

Brassica genus
artificial selection!
Animal Husbandry / Breeding
artificial selection!
Biotechnology Today
 Genetic Engineering
direct manipulation of DNA
 if you are going to engineer DNA &
genes & organisms, then you need a
set of tools to work with
 this unit is a survey
of those tools…

Our tool kit…
Bioengineering Tool Kit
 Basic Tools
restriction enzymes
 ligase
 plasmids for gene cloning
 gel electrophoresis

 Advanced Tools
PCR
 DNA sequencing
 Southern blotting
 DNA libraries / probes
 microarrays

Cut, Paste, Copy, Find…
 Word processing metaphor…

cut (Ctrl + X)
 restriction enzymes

paste (Ctrl + V)
 ligase

copy (Ctrl + C)
 via plasmids
 bacteria
 transformation
 via PCR

find (Ctrl + F)
 Southern blotting
 probes
Cutting DNA
 Restriction enzymes
restriction endonucleases
 discovered in 1960s
 evolved in bacteria to cut up foreign
DNA (“action restricted to foreign DNA”)

 protection against viruses
& other bacteria
 bacteria protect their
own DNA by methylation
& by not using the base
sequences recognized
by the enzymes in their
own DNA
Paste DNA
 Sticky ends allow:

H bonds between
complementary
bases to anneal
 Ligase

enzyme “seals”
strands
 bonds sugar-
phosphate bonds
 covalent bond of
DNA backbone
Biotech Use of Restriction Enzymes
GAATTC
CTTAAG
Sticky ends (complementary
single-stranded DNA tails)
GAATTC
CTTAAG
Restriction enzyme
cuts the DNA
AATTC
G
Add DNA from another
source cut with same
restriction enzyme
G
CTTAA
AATTC
G
G
AATTC
CTTAA G
DNA ligase
joins the strands.
Recombinant DNA molecule
DNA
GAATTC
CTTAAG
Application of Recombinant DNA
 Combining sequences of DNA from
2 different sources into 1 DNA molecule

often from different species
 human insulin gene in E. coli (humulin)
 frost resistant gene from Arctic fish in
strawberries
 “Roundup-ready” bacterial gene in soybeans
 BT bacterial gene in corn
 jellyfish glow gene in
Zebra “Glofish” – GFP!
1961, 1994 | 2008
Development of GFP
 Shimomura, Chalfie, Tsien

discovery, isolation, and purification of
GFP and many fluorescent analogs
Osamu Shimomura
Martin Chalfie
Roger Tsien
Cut, Paste, Copy, Find…
 Word processing metaphor…
  restriction enzymes
paste
 ligase

cut



copy
 plasmids
 bacteria
 transformation
 PCR (chapter 11)


find

 Southern blotting
 probes
Plasmids
Trust me…
this will be
important!
Plasmids
 Plasmids

small supplemental circles of DNA
 5000 - 20,000 base pairs
 self-replicating

carry extra genes
 2-30 genes

can be exchanged between bacteria
 bacterial ‘sex’!!
 rapid evolution
 antibiotic resistance

can be imported
from environment
Transformation
 Bacteria are opportunists

pick up naked foreign DNA wherever it
may be hanging out
 have surface transport proteins that are
specialized for the uptake of naked DNA
import bits of chromosomes from other
bacteria
 incorporate the DNA bits into their own
chromosome

 express new gene
 form of recombination
Who’s
experiment
documented
this?
Swapping DNA
 Genetic recombination by trading DNA
1
arg+
trp-
minimal
media
3
2
argtrp+
Plasmids & Antibiotic Resistance
 Resistance is futile?
1st recognized in
1950s in Japan
 bacterial dysentery
not responding to
antibiotics
 worldwide
problem now

 resistant genes
are on plasmids
that are swapped
between bacteria
Copy DNA
 Plasmids

small, self-replicating
circular DNA molecules
 insert DNA sequence into plasmid
 vector = “vehicle” into organism

transformation
 insert recombinant plasmid into bacteria
 bacteria make lots of copies of plasmid
 grow recombinant bacteria on agar plate
 clone of cells = lots of bacteria
 production of many copies of inserted gene
DNA  RNA  protein  trait
Biotechnology
 Used to insert new genes into
bacteria

example: pUC18
 engineered plasmid used in biotech
antibiotic
resistance gene on
plasmid is used as
a selective agent
Biotechnology
 Used to insert new genes into
bacteria

example: pUC18
 engineered plasmid used in biotech
antibiotic
resistance gene on
plasmid is used as
a selective agent
Selection for Plasmid Uptake
 Ampicillin becomes a selecting agent

only bacteria with the plasmid will grow
on amp plate
all bacteria grow
only transformed
bacteria grow
LB plate
LB/amp plate
Recombinant Plasmid
 Antibiotic resistance genes as a selectable marker
 Restriction sites for splicing in gene of interest
Selectable marker
 Plasmid has both
“added” gene &
antibiotic resistance
gene
 If bacteria don’t pick
up plasmid then “die”
on antibiotic plates
 If bacteria pick up
plasmid then survive on
antibiotic plates
 selecting for successful
transformation
selection
GFP
Gene Cloning
Cut, Paste, Copy, Find…
 Word processing metaphor…
  restriction enzymes
paste
 ligase

cut

  plasmids
  bacteria

copy
 transformation
 PCR (chapter 11)


find

 Southern blotting
 probes
Any Questions?