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Chapter 20
DNA Technology and Genomics
The Challenges of Studying DNA
The size of the DNA poses big
problems.
 Naturally occurring DNA is very long
and particular genes may only
comprise a small portion of the DNA,
maybe 1/100,000 of the chromosome.
 There may only be a small difference in
the surrounding nucleotides.

Help in Overcoming These
Challenges

To make this easier, scientists have
developed methods for gene cloning.
 Genes are cloned to make multiple copies of
the same gene and to produce a protein
product.
 Cloning can be used to endow an organism
with a new metabolic activity.
–

Pest resistance, drought resistance, etc.
We can also insert genes into organisms for
medical purposes.
–
Insulin production by E. coli.
Most Methods for Cloning
DNA Share General Features:

A commonly used method uses
bacteria (E. coli) and their plasmids:
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1. The plasmid is isolated from bacteria.
2. DNA is inserted into it.
3. The plasmid is now recombinant (DNA
from 2 sources).
– 4. The bacteria reproduces forming a
clone of the original cell.
– 5. The foreign (inserted) gene is cloned at
the same time.
Restriction Enzymes
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These are enzymes
that cut DNA molecules
at a limited number of
specific locations.
In nature, these
enzymes help prevent
a bacterial cell from
foreign DNA (from
phages and other
organisms).
Many different
restriction enzymes
have been identified
and isolated.
Restriction Enzymes

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Each restriction enzyme
is very specific and
recognizes a short DNA
sequence known as a
restriction site.
The DNA itself is cut at
specific sites within the
DNA strand.
A bacterial cell will
protect its own DNA from
its own restriction
enzymes by addition of
methyl (-CH3) groups to
A’s and C’s within the
sequences recognized
by these enzymes.
Restriction Enzymes
They recognize sequences 4-8
nucleotides in length.
 Many such sequences occur by
chance throughout the genome, thus a
restriction enzyme will produce a
numerous amount of fragments (called
restriction fragments) when they are
introduced to DNA.

Restriction Enzymes
All copies of a particular DNA molecule
always produce the same DNA
fragments when introduced to the
same restriction enzymes.
 Thus, a restriction enzyme cuts DNA in
a reproducible way.

Restriction Enzymes
The most useful RE’s cleave DNA in a
certain way and produce sticky ends.
 We call them sticky ends because they
combine with other DNA fragments that have
been cut by the same enzyme.
 These fragments usually hydrogen bond
together and then are joined permanently by
DNA ligase which catalyzes the formation of
covalent bonds in the sugar-phosphate
backbones.
 This produces a stable, recombinant DNA
molecule.

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20_03RestrictionEn
zymes_A.mov
Gene Cloning in Plasmids
Genes are cloned in plasmids using a
cloning vector which is the original
plasmid that carries the foreign DNA
into the cell.
 Bacterial plasmids are commonly used
because they are easy to manipulate
and most of the experimentation with
them can be done in vitro.

A Common Method for
Cloning:
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1. Isolation of plasmids from E. coli cells
and DNA from human cells grown in culture.
2. Treat the 2 with the same RE producing
the same sticky ends. Plasmids cut at one
spot, human DNA cut at many.
3. Mix the human and plasmid fragments.
4. Add DNA ligase to permanently fuse the
sticky ends of the plasmid and human DNA.
5. Mix the recombinant plasmids with
bacteria.
6. Plate the bacteria out on selective media
to isolate the recombinants.
Recognizing the Clone
Originally, the bacteria lacked a gene
conferring resistance to something, say
an antibiotic.
 When the bacterium was plated on
growth medium containing the
antibiotic, they would die.
 Those bacteria containing the antibiotic
resistance gene are now able to
survive on the medium containing
antibiotic.

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20_04CloningAGen
e_A.mov
Problems with Cloned DNA
and Expression in the Bacteria
Two different types of cells (prokaryotic
vs. eukaryotic) pose problems for gene
expression.
 The presence of introns in eukaryotic
transcripts and no way for the
prokaryotes to splice them out.

2 Different Cell Types
To overcome this, scientists use
expression vectors that have highly
active prokaryotic promoters just
upstream from the gene to be
expressed.
 The bacterial cell will now recognize
the promoter and express the foreign
DNA linked to the promoter.
 In this way, many eukaryotic genes are
expressed in prokaryotic cells.

The Presence of Introns
Prokaryotes lack splicing machinery,
and the long eukaryotic gene is often
prevented from being expressed in the
bacteria.
 Scientists use cDNA to circumvent the
problem. The cDNA contains only the
exons.

cDNA Synthesis

cDNA is synthesized from mRNA extracted
from the cells.
 Retroviruses make reverse transcriptase and
this is used to make single stranded DNA
molecules from the mRNA.
 mRNA gets enzymatically degraded and
DNA polymerase then synthesizes a second
strand of the DNA.
 The cDNA is modified with REs to ease the
transition into plasmids and then bacterial
cells.
cDNA Synthesis

The cDNA will now be expressed by
the bacterial cell as long as the vector
contains the bacterial promoter and
other control elements necessary for
transcription and translation.
Other Ways to Get Around
Compatibility Problems:
Scientist often use eukaryotic yeasts
and single-celled fungi which are as
easy to grow as bacteria and also
contain plasmids.
 Scientists have also made recombinant
plasmids that combine yeast and
bacterial DNA that can replicate in
either type of cell.

Other Ways to Get Around
Compatibility Problems:
Yeast artificial chromosomes are
another way in which scientists clone
eukaryotic chromosomes.
 Here, the essentials of a eukaryotic
chromosome (an origin for DNA
replication, a centromere, 2 telomeres)
along with the foreign DNA to be
replicated is all that is needed.

Yeast Artificial Chromosomes

These chromosome-like vectors behave
normally in mitosis and clone the foreign
DNA as the cell divides. The YAC is a lot
longer than a plasmid, and it is more likely to
contain the entire gene rather than a portion
of it.
 Eukaryotic cells are desired because
prokaryotic cells cannot modify the proteins
after they have been expressed.
 Sometimes this doesn’t work and an animal
cell may be needed.
PCR
Sometimes scientists want to prepare a
large quantity of DNA when only a
small amount is present.
 To get around this, PCR is used and
can quickly generate a large amount of
DNA from a small amount.

PCR

A 3-step cycle brings about a chain reaction
that produces exponential growth of identical
DNA molecules.
 A double stranded piece of of DNA is
obtained.
–
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A. The solution containing the piece of DNA is
heated so as to denature the DNA and separate
it into single strands.
B. DNA primers (short, single stranded DNA
molecules) are added to the mixture and it is
allowed to cool so the primers anneal to the
cDNA strands.
C. The heat-stable DNA polymerase adds
nucleotides to the primers in the standard 5’-->3’
direction synthesizing the target sequence.
PCR
What makes this process so useful is
its specificity. If a target segment is
identified and a primer made to it, then
only a small amount is really necessary
from the start.
 It is easy to see how quickly a large
amount of DNA can be made: 1, 2, 4,
8, 16, …..
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PCR
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The primer will only replicate the target
segment because this is all they are
able to bind to. After just a few cycles,
a very large amount of the target
segment will be identified.
PCR
DNA cloning in cells remains the best
way to prepare a large quantity of a
gene or DNA segment. PCR can’t be
used to obtain a large quantity of gene
because occasional errors in PCR
replication impose limits on the number
of good copies that can be made.
 Often times though, enough of a
specific DNA fragment can be made to
insert it into a vector and clone it.
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Gel Electrophoresis
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To study DNA,
scientists often use gel
electrophoresis.
Agarose gel is often
used to separate DNA
fragments based on
size, charge, etc., that
have been treated with
a restriction enzyme.
The fibers in the gel
separate out the
fragments; smaller
fragments migrate
further than the larger
fragments.
Gel Electrophoresis

The negatively charged DNA fragments
migrate toward the positive pole of the
electrophoresis box.
Restriction Fragment Analysis
This is a powerful tool scientists use to
analyze differences in the nucleotide
sequences of DNA molecules.
 When the researchers began analyzing
the restriction fragments of non-coding
DNA from individuals, they began
noticing small nucleotide differences on
homologous chromosomes.
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Restriction Fragment Analysis
Treating the DNA with restriction
enzymes and then running the samples
through a gel enable researchers to
produce banding patterns
characteristic of the starting molecule
and the restriction enzyme(s) used to
treat the DNA.
 You will use this analysis to examine
your bacterial chromosome for certain
genes.
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QuickTime™ and a
TIFF (U ncompressed) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (U ncompressed) decompressor
are needed to see this picture.
Restriction Fragment Length
Polymorphisms
When non-coding regions of DNA were
treated with restriction enzymes and
banded, scientists discovered
differences in non-coding regions on
homologous chromosomes.
 These were given the name restriction
fragment length polymorphisms.
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Restriction Fragment Length
Polymorphisms
These serve as genetic markers of
non-coding DNA that appears near a
particular locus in a genome.
 There are many RFLP variants within a
population.
 RFLP data is often used in crime
investigations because the likelihood
that two individuals will have the same
banding patterns are miniscule at best.
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Scientists use a Southern Blot to
Analyze RFLPs or Regular DNA.
So, How Are They Analyzed?
Follow the link: whfreeman.com
Why is This Important?

It is used in crime
scene
investigations and
paternity suits all
the time.
Why is This Important?
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Scientists use the
information to
determine if a
person has a
particular disease.
Why is This Important?

Scientist can also use RFLP information to
determine the likelihood of inheriting a
certain genetic disease.
HGP Project

Was started in 1990 and was completed in
2003. The goal was to map the entire
human genome.
 It proceeded in 3 stages:
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Making a genetic (linkage) map.
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Making a physical map.
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Using genetic markers to determine the order of genes.
Determining the distance between genes.
DNA sequencing.
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Determining the exact nucleotide sequence of the
genes.
DNA Sequencing
Became the biggest drawback. There
wasn’t a fast way to sequence the
genes. The initial challenge was to
develop new technologies to quickly
sequence the DNA.
 Frederick Sanger developed the first
method to sequence the genes called
the dideoxy chain-termination method.

The Dideoxy ChainTermination Method
A set of complementary DNA strands are
synthesized from an original DNA strand.
 Each strand starts with the same primer
and ends with a modified nucleotide that
is fluorescently labeled and lacks a
hydroxyl group; it is called a
dideoxyribonucleotide.
 The dideoxyribonucleotide terminates the
growing DNA strand because it lacks a 3’OH group.

The Dideoxy ChainTermination Method
In the newly synthesized strands, each
nucleotide position along the original
sequence is represented by strands
ending at that point with the
complementary ddNTP.
 Each type of ddNTP, tagged with a
distinct fluorescent label, identifies the
ending nucleotides of the new strands,
ultimately revealing the sequence of
the DNA.
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3 Main Steps of the Dideoxy
Method
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1. The fragment of DNA to be
sequenced is denatured into single
strands and incubated in a test tube
with a primer known to base pair with
the known 3’ end of the template stand,
DNA polymerase, A,T,C,&G and the 4
ddNTP’s which are tagged with a
specific fluorescent molecule.
3 Main Steps of the Dideoxy
Method

2. Synthesis of the new strand starts
at the 3’ end of the primer and
continues until a ddNTP is added at
random. This prevents further
elongation. Eventually, a labeled set of
strands of various lengths is generated
3 Main Steps of the Dideoxy
Method
3. The labeled strands in the mixture
are separated by passage through a
polyacrylamide gel in a capillary tube;
the shorter the strands move through
faster than the larger ones.
 A fluorescent detector can sense the
color of each tag as the strands come
through. Strands that differ in as little
as 1 nucleotide can be distinguished.
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The Dideoxy Method
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The color of the fluorescent tag on
each strand indicates the identity of
each nucleotide at its end, and the
results can be printed out on a
spectrogram and the sequence, which
is complementary to the template
strand, can then be read from the
bottom to top.
Whole Genome Shotgun
Approach
In 1992, Craig Venter developed the
shotgun method which greatly sped up
the process of gene sequencing.
 The process uses random DNA
fragments and powerful computers to
analyze the data.
 The idea was initially met with
skepticism.
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Whole Genome Shotgun
Approach
What is essentially happening is the
piece of DNA is incubated with a
variety of restriction enzymes and then
cloned in plasmid of phage vectors to
produce large numbers of these
fragments.
 Then, each fragment is sequenced and
a computer program analyzes the
fragments relative to each other to
determine the sequence.

Whole Genome Shotgun
Approach
His approach sequenced nearly the
entire genome in about 3 years.
 Using the Sanger method, the HGP
took nearly 11 years.
 Originally, Venter and his colleagues
were accused to stealing the
information made available by the HGP
to sequence the genome, but it is
generally understood now that the
Shotgun approach is a good method.
