Chapter 11

FlexArt
Labels and leader lines are editable on every image in this
PowerPoint.

To get started, select an image from within the PowerPoint file and
simply click on any of the labels and edit or delete to your preference.

PowerPoint Tips: Refer to the Microsoft Help feature for specific

questions about PowerPoint.

Business Unit Tag line (optional) Copyright © McGraw-Hill Education. Permission required for reproduction or display. 1
Fig. 11.1 Genes or gene products can be silenced/expressed (epigenetics):
TRANSCRIPTION TRANSLATION

Nucleus Cytoplasm Post-replication modification:

In prokaryotes, RNA Ribosome Via histone methylation/acetylation
transposable elements Protein

(also known as
transposons, insertion
sequences, or jumping
jeans) are genes
flanked by palindromic P
sequences, or inverted
DNA
Post-transcriptional modification:
98.5% of DNA does not code for proteins
- Regulatory RNA genes (anti-sense)
- siRNA (small inhibitory RNA):
DNA
- small bits of non-coding RNA
C
G

repeats, that code for

P
target mRNA for cleavage
P

enzymes that can cut G

C
P - miRNA (microRNA)
the transposon out of a P
T
- Prevents translation
chromosome and paste
P

P
it in another part
to create genetic
- Introns
G
C P
- Remnants of ancestral genes
P C

variety. P
G
T
P
- Pseudogenes (imperfect copies and
A
G
C
P
TEs—44% of DNA, 99.95% silenced)
In eukaryotes, A - Gene regulatory sequences
transposons are - Promotor, operator/silencer, enhancer
silenced via
epigenetics.

Post-translational modification:
- Phosphorylation by protein kinase
- Addition/removal of molecular groups
Inverted repeat = GODDAMMADDOG
Double-stranded

Short hairpin

Small interfering
Small inhibitory

RNA-induced silencing
complex, or RISC

100%
Complementary
RNA-induced silencing complex, or RISC

Like siRNA Unlike siRNA

Fig. 11.2 Copyright © McGraw-Hill Education. Permission required for reproduction or display.

Human
cell

1 Acquire source DNA

DNA from human cell 2 Obtain a plasmid

Plasmid (circle of DNA)

3 Make recombinant DNA

Restriction enzymes cut
DNA at specific sequence
(GAATTC in this case)
AA T T C
AAT T C

Segment containing
gene of interest

Mix donor DNA with

plasmid DNA to construct
recombinant plasmid

Genetically modified bacteria

can be used to make:
4 Insert the recombinant DNA - human insulin to treat
into a recipient cell
diabetes.
- blood clotting factors to
treat hemophilia.
- fertility hormones for
humans.
- amino acid phenylalanine
Recombinant Transgenic bacterium
used in the artificial
plasmid containing human DNA
sweetener aspartame.
Fig. 11.3

Copyright © McGraw-Hill Education. Permission required for reproduction or display.

1 Recombinant Ti plasmid

Agrobacterium
When the transgenic cell divides, each
Chromosome daughter cell receives the herbicide resistance
Herbicide gene. The resulting tobacco plant is transgenic.
resistance gene

2 Infection Herbicide resistance gene

3
Cell Cell
division division

Chromosome

Unaltered tobacco cell Transgenic tobacco cell

 DNA Replication Requires a Primer
Remember, RNA was the original code before
DNA. Thus, RNA polymerase had to be able to
add the first nucleotide without a “hook.”
Primase creates the “hook” for DNA polymerase.
NEEDS A PRIMER.
What is a primer?
An RNA primer must be
synthesized by an enzyme called
primase, which is a type of RNA
polymerase, before DNA
replication can occur.
Why is a primer needed?
The synthesis of a primer is
necessary because the enzymes that
synthesize DNA, which are
called DNA polymerases, can only
attach new DNA nucleotides to an
existing strand of nucleotides.
What if there was no primer?
Without a 3’ hydroxyl group
to act as a “hook," a new
nucleotide cannot be added to
an existing chain.
Topoisomerases are enzymes that
participate in the over winding or
underwinding of DNA.
Synthesis of Okazaki fragments requires RNA primers attaching ahead on the lagging strand.
Why is this an issue?
During chromosome replication, the enzymes that duplicate DNA cannot continue their duplication all
the way to the end of a chromosome, so in each duplication the end of the chromosome is shortened.

How do we keep from losing valuable genes?

A telomere is a region of repetitive nucleotide sequences at each end of a chromosome, which
protects the end of the chromosome from deterioration or from fusion with neighboring chromosomes.

What happens to the telomeres after each cell division?

Over time, due to each cell division, the telomere ends become shorter. They are replenished by an
enzyme, telomerase reverse transcriptase.

But once the telomeres run too short, the cell stops dividing (recall: checkpoints ensure all DNA is
replicated before proceeding).

Why are women of advanced maternal age prone to having children with genetic defects?
A woman is born with all the eggs that she will ever ovulate (release from ovary into fallopian tube for
fertilization).
 Primer Nucleotides
In Sanger sequencing, the target In sequencing, we
DNA (900 base pairs or less) is introduce a pre-made
copied many times, making DNA primer. Unknown DNA sequence
fragments of different lengths. Replication enzymes
Fluorescent “chain terminator” including DNA
polymerase
nucleotides mark the ends of the Lots of dATP, dTTP, dCTP, dGTP 1 Each solution contains the unknown DNA sequence, replication
enzymes, primers, normal nucleotides (A, C, T, and G), and a small
fragments and allow the amount of one type of labeled “terminator” nucleotide (A*, C*, T*, or G*).
sequence to be determined. A little bit of ddATP, ddTTP, ddCTP, ddGTP Terminator A Terminator C Terminator T Terminator G
added added added added

Dideoxy, or chain-terminating, versions of all four

nucleotides (ddATP, ddTTP, ddCTP, ddGTP), are each
labeled with a different color of dye (linked to the base).
2 Replication occurs, producing fragments of complementary copies
of the unknown sequence.
ATG*
A* ATGC*
Without a 3’ hydroxyl group ATGCGCA* ATGCGC*
AT* ATGCG*
ATGCGCAT* ATGCGCATG*
to act as a “hook," a new
nucleotide cannot be added to
an existing chain. Capillary
3 Sequencing machine uses
capillary gel electrophoresis
to sort fragments by size. Detector

Laser

4 Laser, detector, and computer

generate readout of DNA sequence.

TTTGATTATCTCCAACAAAGTTAGGG
REPLICATION PRODUCES COMPLEMENTARY FRAGMENTS OF THE UNKNOWN SEQUENCE
Primer Nucleotides

Unknown DNA sequence

Replication enzymes
including DNA
polymerase

1 Each solution contains the unknown DNA sequence, replication

enzymes, primers, normal nucleotides (A, C, T, and G), and a small
amount of one type of labeled “terminator” nucleotide (A*, C*, T*, or G*).

Terminator A Terminator C Terminator T Terminator G

added added added added

2 Replication occurs, producing fragments of complementary copies

of the unknown sequence.
ATG*
A* ATGC* AT* ATGCG*
ATGCGCA* ATGCGC* ATGCGCAT* ATGCGCATG*

Capillary
3 Sequencing machine uses
capillary gel electrophoresis
to sort fragments by size. Detector

Laser

4 Laser, detector, and computer

generate readout of DNA sequence.

TTTGATTATCTCCAACAAAGTTAGGG
 …but without the cell

rapidly replicates DNA sequences without the use of living

organisms
 1 DNA polymerase , nucleotides, primers, and
PCR, by creating 2n copies where n = number of target DNA sequence are combined.

thermal cycles, ensures a ddNTP is incorporated at Taq DNA polymerase

every base position…
Nucleotides

Primers
Steps in a thermal cycle:
Taq is a heat
1. Ingredients are combined. tolerant DNA
2. The mixture is first heated to denature the template polymerase
DNA (separate the strands). Target sequence from a
3. The mixture is then cooled so that the primer can bind to Round 1: produces prokaryote
the single-stranded template. 2 copies
which lives in
4. Once the primer has bound, the temperature is raised 2 Temperature is raised, causing hot springs.
again, allowing DNA polymerase to synthesize new the strands to separate.

DNA starting from the primer.

5. DNA polymerase will continue adding nucleotides to
the chain until it happens to add a dideoxy nucleotide 3 Temperature is lowered,
and primers from the solution
instead of a normal one. At that point, no further attach to the target sequence.
nucleotides can be added, so the strand will end with the
dideoxy nucleotide. 4 DNA polymerase
finishes replicating DNA,
6. Repeat step 2-5, using the copies as a template. yielding two copies of the
target sequence.

This process is repeated in a number of cycles. By the time

the cycling is complete, it’s virtually guaranteed that a
dideoxy nucleotide will have been incorporated at every
single position of the target DNA in at least one reaction. Round 2: produces
4 copies
That is, the tube will contain fragments of different lengths,
ending at each of the nucleotide positions in the original
DNA (see figure below). The ends of the fragments will be Round 3: produces
labeled with dyes that indicate their final nucleotide. 8 copies
 Sequencing machine uses capillary gel electrophoresis to sort fragments by size.

After the reaction is done, the fragments are

run through a long, thin tube containing a
gel matrix in a process called capillary gel
electrophoresis.

Short fragments move quickly through the

pores of the gel, while long fragments move
more slowly. As each fragment crosses the
“finish line” at the end of the tube, it’s
illuminated by a laser, allowing the attached
dye to be detected.

The smallest fragment (ending just one

nucleotide after the primer) crosses the
finish line first, followed by the next-
smallest fragment (ending two nucleotides
after the primer), and so forth.

Thus, from the colors of dyes registered one

after another on the detector, the sequence
of the original piece of DNA can be built up
one nucleotide at a time.

The data recorded by the detector consist of
a series of peaks in fluorescence intensity,
as shown in the chromatogram above. The
DNA sequence is read from the peaks in the
chromatogram.
Laser, detector, and computer
generate readout of DNA sequence.
Electrophoresis takes advantage of charge
to separate molecules based on size.
 Next-generation sequencing

•Highly parallel: many sequencing reactions take

place at the same time
•Micro scale: reactions are tiny and many can be done
at once on a chip
•Fast: because reactions are done in parallel, results
are ready much faster
•Low-cost: sequencing a genome is cheaper than with
Sanger sequencing
•Shorter length: reads typically range from 50 to 700
nucleotides in length

Conceptually, next-generation sequencing is kind of like running a very large number of tiny Sanger sequencing
reactions in parallel. Thanks to this parallelization and small scale, large quantities of DNA can be sequenced
much more quickly and cheaply with next-generation methods than with Sanger sequencing. For example, in
2001, the cost of sequencing a human genome was almost one million dollars. In 2015, it was just $1245!
 The number of STRs at a given locus varies from one person to the next, so sequencing them is useful in
DNA fingerprinting. They are short 2-10 base pairs and dispersed evenly throughout the genome.
1 Extract DNA from a diploid cell from 2 Amplify STR sequences using PCR. Reference profile

Relative fluorescence
each of the three suspects. N= number of repeats
2000 78

1500 9
6 10
1000
500
Chromosome 5
(homologous pair) 100 120 140
Number of base pairs

3 Sequence the amplified DNA to determine the number of repeats and generate each man’s profile.
The reference profile in the upper right corner shows all possible alleles for this STR site.
Short tandem repeat (STR), 7 copies DNA profiles
7
Genotype:
7, 9 9
C C A G A T A G A T A G A T A G A T A G A T A G A T A G A T C G A C C A G

C C A G A T A G A T A G A T A G A T A G A T A G A T A G A T A G A T A G A T C G A C C A
Man 1

8
Genotype:
6, 8
C C A G A T A G A T A G A T A G A T A G A T A G A T A G A T A G A T C G A C C A G 6

C C A G A T A G A T A G A T A G A T A G A T A G A T C A A C C A G
Man 2

Genotype:
C C A G A T A G A T A G A T A G A T A G A T A G A T C A A C C A G
6,10
6 10

C C A G A T A G A T A G A T A G A T A G A T A G A T A G A T A G A T A G A T A G A T C G
Man 3

Let’s say 1% of people have the same number of allelic repeats at locus A as you, 1% at locus B, 1% at locus C, etc. Using the
product rule, you will quickly see that, for 10 loci, the % of individuals who match you for all loci would be (.01)^10 or 1 in a
trillion … there were only 100 billion humans ever to have walked the earth! Indeed, we have a finger print.
 Copyright © McGraw-Hill Education. Permission required for reproduction or display.
Fig. 11.7
STR Locus Genotypes
Chromosome Number of DNA Man 1 Man 2 Man 3
(Locus name) possible from
repeats crime
scene

2 (TPOX) 4–16 10, 11 9, 10 10, 11 8, 8

3 (D3S1358) 8–20 17, 19 19, 19 17, 19 15, 16
4 (FGA) 13–51 20, 24 22, 24 20, 24 21, 23
5 (D5S818) 6–18 6, 8 7, 9 6, 8 6, 10
5 (CSF1PO) 5–16 7, 10 10, 11 7, 10 6, 12
7 (D7S820) 5–16 13, 14 8, 12 13, 14 9, 10
8 (D8S1179) 7–20 11, 12 13, 14 11, 12 13, 13
11 (TH01) 3–14 7, 7 9, 12 7, 7 8, 9
12 (vWA) 10–25 15, 12 17, 19 15, 12 18, 19
13 (D13S317) 5–17 10, 10 14, 14 10, 10 9, 13
16 (D16S539) 4–16 9, 13 10, 11 9, 13 9, 9
18 (D18S51) 7–40 15, 21 20, 21 15, 21 14, 18
21 (D21S11) 12–41 30, 32 16, 18 30, 32 29, 30
a.
DNA exonerations

25
20
in the U.S.

15
10
5
0
1990 1995 2000 2005 2010
b.
Fig. 11.8

Copyright © McGraw-Hill Education. Permission required for reproduction or display.

a.

b.
a: ©Congressional Quarterly/Getty Images; b: ©Getty Images
 Embryonic stem cells
(develop into every cell type in the body)

Fertilized Embryonic
egg stem cells

Recall: gene expression can be modified to differentiate cells Early embryo

(5–6 days after
fertilization)

Via epigenetic modification: Tests of embryonic stem

Histone methyltransferase cells have generally shown
- Activate gene expression very poor results. In contrast,
- Inhibit gene expression adult stem cells have already
Histone acetyltransferase been successfully used in
- Activate gene expression therapies
Neurons Muscle cells Red blood cells
a.
In biology, oligopotency is the ability of progenitor cells to
Adult stem cells
differentiate into a few cell types. It is a degree of potency. (develop into a subset of cell types)

Totipotent cells: can form all the cell types in a body,

plus the extraembryonic, or placental, cells. Bone marrow
Embryonic cells within the first couple of cell
divisions after fertilization are the only cells that are Adult stem cells
totipotent. (two types)

Pluripotent cells: can give rise to all of the cell types Stem cells in adults are
that make up the body; embryonic stem cells are more differentiated.
considered pluripotent.
one cell type, but are more limited than pluripotent Stem cells can
cells; adult stem cells and cord blood stem cells are be coaxed to
considered differentiate
Multipotent cells can develop into more than into the needed Red and white Fat and bone cells

multipotent. specific cell blood cells (among others)

b.
type.
How does DNA profiling detect genetic
differences between individuals?
A. uses short tandem repeats and other
variable parts of the genome
B. uses shared genetic sequences
C. uses entire genomes
D. uses detection of non-AGCT bases
E. uses only coding regions of the genome
Page 207

This is most similar to what kind of reproduction?

 Dolly showed that genes in the nucleus of such a mature differentiated somatic cell are still capable of reverting
to an embryonic totipotent state [via epigenetic modification], creating a cell that can then go on to develop
into any part of an animal.
Normally , somatic cells do not specialize into stem
cells.
DNA technology, here, is being used to turn genes on.
Cells from animal 1 Obtain donor Embryo
to be cloned Cell nucleus.

Egg cell
3 Fuse 4 Cell divides 5 Transfer embryo
Egg denucleated to form to surrogate 6 Embryo develops
donor 2 Remove nucleus Denucleated egg with embryo. mother’s uterus. into Dolly.
from egg and egg cell nucleus.
discard.

Dolly was cloned by Keith Campbell, Ian Wilmut, and colleagues at the Roslin Institute in
Scotland in 1996. Wilmut stated, “Dolly is derived from a mammary gland cell and we couldn't
think of a more impressive pair of glands than Dolly Parton’s.”
Used to search for specific sequences of DNA

various disease-causing alleles can be detected using DNA technology

Fluorescent label
Cystic fibrosis
allele
T C T A T T C C G A G
Labeled probe complementary
to cystic fibrosis allele
Sequence #1 (complementary to probe) LM 4 µm

Binding occurs;
cystic fibrosis
T C T A T T C C G A G allele detected
A A GCA G A T A A G G C T C A T C G

Sequence #2 (not complementary to probe)

No binding;
cystic fibrosis
T C T A T T C C G A G allele not
detected
T A T GC A T C G A T T A T GA T A C
Fig. 11.12

a.
LM 25 µm

b.
Fig. 11.13 Use of transduction to create a recombinant chromosome.
Mutation
CFTR gene

Chromosome 7

1 Cystic fibrosis occurs in people with mutations Abnormal

Gene therapy in the CFTR gene; lung cells produce abnormal CFTR protein
CFTR proteins.
might someday
be used to treat
many genetic
disorders by
removing faulty
genes from
2 Healthy version
somatic cells 4 Lung cells produce Normal of CFTR gene
normal CFTR proteins. is placed inside
and replacing CFTR protein
viruses.
them with
functional gene
copies.

3 Patient inhales
viruses, which carry
healthy CFTR genes
into multiple lung cells.
Page 211
Applications of DNA technology:
- detection of genetic illnesses
- replacement of faulty copies of genes
- production of genetically modified crops
with greater nutritional value
- DNA profiling criminal suspects
- Use of bacteria to produce human
proteins like insulin, clotting factors,
fertility hormones, etc
Risks of DNA technology:
- creating too much of a cellular product
- insertion into an incorrect locus
- triggering cancer
- triggering unwanted immune
responses
- ethical considerations (i.e. who gets
health insurance, gene doping in
athletics)
Fig. 11.B Here we have production of genetically modified crops with greater fertility, nutrition, etc

100 1200

90 1100
1000
80

Number of seeds per plant

900
Seed germination rate (%)

70
800
60 700
50 600

40 500
400
30
300
20
200
10 100
0 0
Normal Weeds with Normal Weeds with
weeds booster weeds booster
a. (control) gene b. (control) gene
Fig. 11.14 Copyright © McGraw-Hill Education. Permission required for reproduction or display.

Technology or Tool Definition

Restriction enzyme Protein that cuts double-stranded

DNA at a specific base sequence

Genetic material that has been cut

Recombinant DNA with restriction enzymes and spliced
with DNA from other sources

Transgenic organism An individual with

recombinant DNA

Determines the nucleotide

DNA sequencing
sequence of DNA fragments

PCR (polymerase Amplifies DNA in a test tube using

chain reaction) the cell’s replication machinery

Uses DNA sequencing and PCR to

DNA profiling detect genetic differences among
individuals
Cells found in embryos and some
Stem cells adult tissues that can give rise to
other cell types

Cloning Makes an identical copy

of an organism

A type of cloning that combines a

nucleus taken from one individual’s
Somatic cell
body cell with a denucleated egg cell
nuclear transfer
from another individual to produce
the first cell of a new organism
A single-stranded sequence of DNA,
labeled with a radioactive isotope
DNA probe or fluorescent tag, used to detect the
presence of a known sequence of
nucleotides
Uses PCR and DNA probes to detect
Preimplantation
genetic diseases in embryos that might
genetic diagnosis
later be implanted in a woman’s uterus
Uses PCR and DNA probes to detect
Genetic testing genetic diseases in fetuses,
newborns, children, and adults

Gene therapy Employs viruses to insert healthy

genes into cells
Fig. 11.15 Copyright © McGraw-Hill Education. Permission required for reproduction or display.
Stem cells Cloning Genetic testing
and

Preimplantation
genetic
diagnosis (PGD)
can develop into produces copies
any type of of an organism’s

rely on

Cell DNA probes

contains highlight a
sequence of

DNA

receives
recombinant identifies a
person based on

Transgenic DNA profiling

organism
adds determines the makes
functional nucleotides in copies of

Man 1 Man 2

DNA Polymerase
Gene therapy chain reaction
sequencing (PCR)

TCTCCAACAAAG