Colonie High AP Biology
DeMarco/Goldberg
DNA Sequencing
 Sanger method
determine the base sequence
of DNA
 dideoxynucleotides

 ddATP, ddGTP, ddTTP, ddCTP
 missing O for bonding of next
nucleotide
 terminates chain
Chapter 17 – 18
Biotechnology:
Genomics & DNA Technology
DNA Sequencing
 Sanger method
Fred Sanger
1
synthesize
complementary DNA
strand in vitro
 in each tube:
This was his 2nd Nobel Prize!!

 “normal” N-bases
 dideoxy N-bases
 ddA, ddC, ddG, ddT
 DNA polymerase
 primer
 buffers & salt


1st was in 1958 for the
structure of insulin
2
3
4
2
Advancements to Sequencing
 Fluorescent tagging

1978 | 1980
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
Colonie High AP Biology
Raw Genome Data
DeMarco/Goldberg
Automated Sequencing Machines
 Really BIG labs!
Human Genome Project
 U.S government project

begun in 1990

DOE & NIH
 estimated to be a 15 year project
 initiated by Jim Watson
 led by Francis Collins

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.
1. Cut DNA from entire chromosome
into small fragments and clone.
2. Sequence each segment & arrange
based on overlapping nucleotide
sequences.
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
Human Genome Project
On June 26, 2001, HGP published the “working
draft” of the DNA sequence of the human genome.
3. Assemble DNA sequence
using overlapping sequences.
How does our genome stack up?
Organism
Human (Homo sapiens)
Historic Event!
blueprint
of a human
 the potential to
change science
& medicine

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
Colonie High AP Biology
GenBank
DeMarco/Goldberg
Organizing the Data
Database of
genetic
sequences
gathered
from
research
Publicly
available!
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.
Colonie High AP Biology
Transposons
 Barbara McClintock

DeMarco/Goldberg
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.
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
Most of these
transposons are
retrotransposons,
transposable
elements that move
within a genome by
means of RNA
intermediate,
transcript of the
retrotransposon
DNA.
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
Colonie High AP Biology
Biotechnology
 Genetic manipulation of organisms is
DeMarco/Goldberg
Evolution & Breeding of Food Plants
not new

humans have been doing this for
thousands of years
 plant & animal breeding
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

Animal Husbandry / Breeding
Brassica genus
artificial selection!
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…

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


Our tool kit…
Colonie High AP Biology
Cut, Paste, Copy, Find…
 Word processing metaphor…

cut (Ctrl + X)
 restriction enzymes

paste (Ctrl + V)

copy (Ctrl + C)
 ligase
DeMarco/Goldberg
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
 via plasmids
 bacteria
 transformation
 via PCR

 bacteria protect their
own DNA by methylation
& by not using the base
sequences recognized
by the enzymes in their
own DNA
find (Ctrl + F)
 Southern blotting
 probes
Paste DNA
 Sticky ends allow:

H bonds between
complementary
bases to anneal
Biotech Use of Restriction Enzymes
GAATTC
CTTAAG
Sticky ends (complementary
single-stranded DNA tails)
 Ligase

enzyme “seals”
strands

often from different species
AATTC
G
G
CTTAA
AATTC
G
G
AATTC
CTTAA G
DNA ligase
joins the strands.
Recombinant DNA molecule
2 different sources into 1 DNA molecule
DNA
Restriction enzyme
cuts the DNA
Add DNA from another
source cut with same
restriction enzyme
 bonds sugarphosphate bonds
 covalent bond of
DNA backbone
Application of Recombinant DNA
 Combining sequences of DNA from
GAATTC
CTTAAG
GAATTC
CTTAAG
1961, 1994 | 2008
Development of GFP
 Shimomura, Chalfie, Tsien

discovery, isolation, and purification of
GFP and many fluorescent analogs
 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!
Osamu Shimomura
Martin Chalfie
Roger Tsien
Colonie High AP Biology
DeMarco/Goldberg
Cut, Paste, Copy, Find…
 Word processing metaphor…
Plasmids
cut
 restriction enzymes
paste
  ligase


copy
 plasmids

 bacteria
 transformation
 PCR (chapter 11)


find
 Southern blotting

 probes
Plasmids
 Plasmids

Transformation
 Bacteria are opportunists
small supplemental circles of DNA

 5000 - 20,000 base pairs


 have surface transport proteins that are
 self-replicating
carry extra genes
 2-30 genes
specialized for the uptake of naked DNA
import bits of chromosomes from other
bacteria
 incorporate the DNA bits into their own
chromosome

can be exchanged between bacteria
 bacterial ‘sex’!!
 rapid evolution
 express new gene
 antibiotic resistance

 form of recombination
can be imported
from environment
Swapping DNA
 Genetic recombination by trading DNA
1
arg+
trp-
pick up naked foreign DNA wherever it
may be hanging out
3
Plasmids & Antibiotic Resistance
 Resistance is futile?
1st recognized in
1950s in Japan
 bacterial dysentery
not responding to
antibiotics
 worldwide
problem now

2
argtrp+
 resistant genes
minimal
media
are on plasmids
that are swapped
between bacteria
Colonie High AP Biology
DeMarco/Goldberg
Copy DNA
 Plasmids

Biotechnology
 Used to insert new genes into
bacteria
small, self-replicating
circular DNA molecules

example: pUC18
 engineered plasmid used in biotech
 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
antibiotic
resistance gene on
plasmid is used as
a selective agent
DNA  RNA  protein  trait
Biotechnology
Selection for Plasmid Uptake
 Ampicillin becomes a selecting agent
 Used to insert new genes into
bacteria


example: pUC18
only bacteria with the plasmid will grow
on amp plate
 engineered plasmid used in biotech
antibiotic
resistance gene on
plasmid is used as
a selective agent
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
all bacteria grow
only transformed
bacteria grow
LB plate
LB/amp plate
Gene Cloning