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Transcript
Chapter 6
Biotechnology:
DNA Technology & Genomics
AP Biology
2005-2006
1
The BIG Questions…
 How can we use our knowledge of DNA to:
diagnose disease or defect?
 cure disease or defect?
 change/improve organisms?

 What are the techniques & applications of
biotechnology?

Biotechnology
 The use of living organisms (or substances
from living organisms) for practical purposes
AP Biology
2005-2006
2
Biotechnology
 Use of organisms
to create a desired
product is not new

humans have
been doing this
for thousands of
years
 Alcohol
fermentation (for
brewing beer and
food preservation)
AP Biology
2005-2006
3
Biotechnology
 Agriculture

Plant and animal breeding
 Selective breeding – altered the genomes of
crops by breeding them with other plants.
AP Biology
2005-2006
4
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 chapter is a survey
of those tools…

AP Biology
2005-2006
5
Bioengineering Tool kit
 Basic Tools




restriction enzymes
ligase
plasmids / cloning
DNA databases / probes
 Advanced Tools





AP Biology
PCR
DNA sequencing
gel electrophoresis
Southern blotting
microarrays
2005-2006
Isolating DNA
Before DNA can be manipulated, it needs to be
isolated from cells.
1. 1. Cell membranes are disrupted


use a detergent
Salt used to neutralize lipids
2. 2. DNA precipitation


ethanol used to dehydrate and aggregate DNA
Salt used to neutralize phosphate groups in DNA
3. 3. DNA isolation / storage
AP Biology
DNA Isolation
AP Biology
1. Cut DNA
 To study the functions
of individual genes,
molecular biologists will
cut desired genes out of
a genome
 place the gene into
bacterial plasmid
 produce recombinant
DNA

AP Biology
2005-2006
1. Cut DNA
 treat isolated DNA
with restriction
enzymes
restriction
endonucleases
 Evolved in bacteria

 Immune System
 protection against
viruses
& other bacteria
AP Biology
2005-2006
Restriction enzymes - Action
 cut DNA at specific sequences
Restriction (recognition) site
 Are usually 4-8 bp in length
 produces protruding ends
sticky ends that contain DNA
nucleotides that lack
complementary bases
 Some do not produce


CTGAATTCCG
GACTTAAGGC
CTG|AATTCCG
GACTTAA|GGC


protruding ends

blunt ends
AP Biology
2005-2006
11
Restriction Enzymes
 Many different
enzymes
 named after
organism
they are
found in
 EcoRI,
HindIII,
BamHI,
SmaI
AP Biology
2005-2006
Restriction Enzyme Cutting
sticky ends – enzyme digests (cuts) to make
overhangs
EcoRI
5’ G A A T T C 3’
3’ C T T A A G 5’
5’ G 3’
5’ A A T T C 3’
3’ C T T A A 5’
3’ G 5’
5’ overhang
AP Biology
Restriction Enzyme Cutting
PstI
5’ C T G C A G 3’
3’ G A C G T C 5’
5’ C T G C A 3’
3’ G 5’
AP Biology
5’ G 3’
3’ A C G T C 5’
3’ overhang
2. Paste DNA
 Sticky ends allow:

H bonds between
complementary
bases to anneal
 DNA Ligase

enzyme “seals”
strands
 bonds sugar-
phosphate
backbone
together
 Condensation
reaction
AP Biology
2005-2006
15
DNA Ligase
 T4 DNA ligase
originated in T4
bacteriophages
 used to chemically
join two blunt ends
of DNA together

AP Biology
Biotech use of restriction enzymes
GAATTC
CTTAAG
Sticky ends (complementary
single-stranded DNA tails)
GAATTC
CTTAAG
DNA
Restriction enzyme
cuts the DNA
AATTC
G
G
CTTAA
Add DNA from another
source cut with same
restriction enzyme
AATTC
G
G
AATTC
CTTAA G
DNA ligase
joins the strands.
AP Biology
Recombinant DNA molecule
GAATTC
CTTAAG
2005-2006
17
 Exercise 1
AP Biology
2005-2006
Cut, Paste, Copy, Find…
 Word processing metaphor…

1. cut
 Isolate desired DNA
 restriction enzymes

2. paste
 ligase

3. copy
 plasmids
 bacteria
 transformation
 PCR

AP Biology
4. find
 Southern blotting / probes
2005-2006
19
Remember…
 Prokaryotic genomes (e.g. bacteria)
contain
Chromosome
 Plasmids

Chromosome
Plasmid
AP Biology
2005-2006
Plasmid pBR322
 Plasmids

Contain “accessory
genes”
 Antibiotic resistance

Can carry and
express foreign genes
 Plasmids are vectors
 Vehicles by which DNA
can be introduced into
host cells
AP Biology
2005-2006
Plasmid Restriction Maps
 Shows the location of
cleavage sites for many
different enzymes


AP Biology
These maps are used
like road maps to the
DNA molecule
Numbers beside
restriction enzyme
indicates at which base
pair the DNA is cut by
that particular enzyme
2005-2006
Sample Problem:
 Determine the size and
number of fragments that
would be produced if the
plasmid was digested
with:
 EcoRV and Pvu II?
 2 cuts, therefore 2
fragments
 Between sites:
2064-185=1879 bp
 Rest:
4361-1879=2482 bp
AP Biology
2005-2006
Bacterium
Cell containing gene
of interest
Why bacteria?
 Plasmid uptake is easy
 Cheap
 Reproduce rapidly and
Gene of
interest
DNA of
chromosome
Recombinant
DNA (plasmid)
frequently
 Produce multiple
copies of the
recombinant DNA
and protein in a
short amount of time
 Recombinant
DNA and proteins
are kept in large
storage database
to be used later
Recombinate
bacterium
3
Gene of
interest
Copies of gene
Protein expressed
by gene of interest
Protein harvested
Basic
research
on protein
Basic
research
on gene
Gene for pest
resistance inserted
into plants
AP Biology
Plasmid
Bacterial
chromosome
Gene used to alter
bacteria for cleaning
up toxic waste
Protein dissolves
blood clots in heart
attack therapy
Human growth
hormone treats
stunted growth
2005-2006
Protein Databases
Actin Protein
Unnamed Protein
AP Biology
Review: Transformation vs. Recombination
 Transformation

the introduction of foreign DNA (usually
a plasmid) into a bacterial cell
 Recombination

AP Biology
Fragment of DNA
composed of
sequences
originating from at
least two different
sources
2005-2006
3. Copying (Cloning)
TECHNIQUE
In this example, a human gene is inserted into a plasmid from E. coli. The plasmid contains
the ampR gene, which makes E. coli cells resistant to the antibiotic ampicillin. It also contains
the lacZ gene, which encodes -galactosidase. This enzyme hydrolyzes a molecular mimic of
lactose (X-gal) to form a blue product. Only three plasmids and three human DNA fragments
are shown, but millions of copies of the plasmid and a mixture of millions of different human
DNA fragments would be present in the samples.
1 Isolate plasmid DNA and human DNA.
2 Cut both DNA samples with the same restriction
enzyme
3 Mix the DNAs; they join by base pairing.
The products are recombinant plasmids and
many nonrecombinant plasmids.
Figure 20.4
AP Biology
lacZ gene
(lactose
Human
breakdown) cell
Bacterial cell
Restriction
site
ampR gene
(ampicillin
resistance)
Bacterial
plasmid
Gene of
interest
Sticky
ends
Recombinant DNA plasmids
Human DNA
fragments
4
Introduce the DNA into bacterial cells (transoformation).
Recombinant
bacteria
5 Plate the bacteria on agar containing
ampicillin and X-gal (lactose). Incubate until
colonies grow.
Colony carrying nonrecombinant plasmid
with intact lacZ gene
Colony carrying
recombinant plasmid
with disrupted lacZ gene
Bacterial
clone
RESULTS
AP Biology
Only a cell that took up a plasmid, which has the ampR gene, will reproduce and form
a colony. Colonies with nonrecombinant plasmids will be blue, because they can
hydrolyze X-gal. Colonies with recombinant plasmids, in which lacZ is disrupted, will be
white, because they cannot hydrolyze X-gal. By screening the white colonies with a
nucleic acid probe (see Figure 20.5), researchers can identify clones of bacterial cells
carrying the gene of interest.
How do we know if it worked?
 We know cloning worked
if:

Transformation has
occurred
 i.e. there has been plasmid
uptake into the bacteria

Recombination has
occurred
 i.e. foreign genes have
been inserted into the
bacterial plasmid
AP Biology
2005-2006
Selecting for successful transformation
 Antibiotic
resistance genes
as a selectable
marker
selection
 Plasmid has both
“added” gene &
antibiotic
resistance gene
AP Biology
2005-2006
30
Amp Selection
 If bacteria don’t pick up
plasmid

they will not have
antibiotic resistance
die on antibiotic
(amp) plates
 If bacteria pick up
plasmid


survive on antibiotic
(amp) plates
 We have selected only
those colonies that have
undergone successful
transformation
AP Biology
2005-2006
Amp Selection
 Ampicillin is the selecting agent
AP Biology
all bacteria grow
only transformed
bacteria grow
LB plate
LB/amp plate
2005-2006
32
Screening for successful recombination
 Transformed
colonies

AP Biology
have both
recombinant
and nonrecombinant
plasmids
recombinant
non-recombinant
2005-2006
LacZ Screening
LacZ gene codes for β galactosidase
Lactose (or X-gal) is our screening agent
EcoRI
BamHI
inserted
gene
of interest
restriction sites
all in LacZ gene
HindIII
LacZ gene
broken
LacZ gene
lactose  blue color
lactose 
X white color
plasmid
recombinant
plasmid
amp
resistance
AP Biology
amp
resistance
origin of
replication
2005-2006
34
LacZ screening system
 Make sure inserted plasmid is
recombinant plasmid

LacZ gene on plasmid
produces digestive enzyme
 lactose (X-gal)  blue
 blue colonies
insert foreign DNA into
LacZ gene breaks gene
X

X
 lactose (X-gal)  blue
 white colonies

AP Biology
white bacterial colonies
have recombinant plasmid
2005-2006
35
Amp selection & LacZ screening
- gene of interest
- LacZ gene
- amp resistance
LB/amp
AP Biology
LB/amp/Xgal
2005-2006
36
Cut, Paste, Copy, Find…
 Word processing metaphor…
  restriction enzymes
paste
  ligase

cut



copy
 plasmids

 bacteria
 transformation
 PCR

find
 Southern blotting / probes
AP Biology
2005-2006
37
4. Find
 White colonies
could have
recombinant
plasmids

desired and
undesired genes
 How do you find
the conony with the
gene of interest?
AP Biology
2005-2006
But how
do we find
colony with our
gene of interest
in it?
4. Find
recombinant plasmids
inserted into bacteria
AP Biology
gene of interest
bacterial colonies (clones) grown
on LB/amp/Xgal petri plates
39
2005-2006
Locating your gene of interest
 DNA hybridization

find gene in bacterial colony using a probe
 short, single stranded DNA molecule
 complementary to part of gene of interest
 tagged with radioactive P32 or fluorescence

heat treat genomic DNA
 unwinds (denatures) strands

DNA hybridization between probe & denatured DNA
labeled probe
genomic DNA
G A T C A G T A G
C T A G T C A T C
3’
AP Biology
2005-2006
5’
Hybridization
4
1 Cloning
- plate with bacterial
colonies carrying
recombinant plasmids
Locate
- expose film
- locate colony on plate
from film
plate
2
plate + filter
Replicate plate
- press filter paper onto
plate to take sample of
cells from every colony
AP Biology
film
filter
Hybridization
- heat filter paper to
denature DNA
3 - wash filter paper with
radioactive probe
which will only attach
2005-2006
to gene of interest
Classwork/Homework
 Pg. 281 #1-3
 Pg. 282 #6,7,10
 Pg. 287 #16,18
 Pg. 289 #20,21
 Pg. 291 #2-7,10,11(b,c),15,16(a,b),17,19
 Pg. 295 #4,5
AP Biology
2005-2006