CAMPBELL

BIOLOGY TENTH
EDITION

Reece • Urry • Cain • Wasserman • Minorsky • Jackson

Lecture 9
(based on Chapter19)

DNA Technology
The DNA toolbox

a) Biotechnology is the manipulation of organisms or their components to

make useful products
b) The applications of DNA technology affect everything from agriculture,
to criminal law, to medical research
Agriculture
Criminal law
Medical Research
Methods to determine protein structure

a) X ray crystallography
b) Nuclear magnetic resonance (NMR) spectroscopy, which does not
require protein crystallization
c) Bioinformatics uses computer programs to predict protein structure
from amino acid sequences
X ray crystallography

EXPERIMENT

Diffracted
X-rays

X-ray
source X-ray
beam

Crystal Digital detector X-ray diffraction

pattern
 RESULTS

RNA
polymerase II

DNA

RNA
Concept 19.1: DNA sequencing and DNA cloning are valuable tools for
genetic engineering and biological inquiry

a) Genetic engineering is the direct manipulation of genes for practical

purposes
DNA Sequencing

a) Researchers can exploit the principle of complementary base pairing to

determine a gene’s complete nucleotide sequence, called DNA
sequencing
b) The first automated procedure was based on a technique called dideoxy
chain termination sequencing, developed by Sanger
DNA Sequencing

a) This determines the order of nucleotides in a DNA strand, allows

scientists to read the genetic code so they can study the normal versions
of genes.
b) It also allows them to make comparisons between normal versions of a
gene and disease-causing versions of a gene
 (a) Standard sequencing machine

use a single template strand

that is immobilized and
amplified to produce an
enormous number of identical
fragments
(b) Next-generation sequencing machines
Making Multiple Copies of a Gene or Other DNA Segment

a) To work directly with specific genes, scientists prepare well-defined DNA

segments in multiple identical copies by a process called DNA cloning
b) Plasmids are small circular DNA molecules in bacteria.
c) Researchers can insert DNA into plasmids to produce recombinant
DNA
d) Now that the plasmid has been recombined the bacteria will express this
foreign DNA and make many copies of this single gene (gene cloning)
 Bacterium Cell containing gene
of interest
1 Gene inserted
into plasmid
Bacterial Plasmid
chromosome DNA of
Gene of chromosome
Recombinant
interest (“foreign” DNA)
DNA (plasmid)
2 Plasmid put into
bacterial cell

Recombinant
bacterium
3 Host cell grown in culture to form a clone of
cells containing the “cloned” gene of interest

Gene of
interest Protein expressed
from gene of interest

Copies of gene Protein harvested

4 Basic research
Gene for pest resistance Human growth hormone
and various
inserted into plants treats stunted growth
applications

Gene used to alter bacteria Protein dissolves blood clots

for cleaning up toxic waste in heart attack therapy
Cloning vector

a) A plasmid used to clone a foreign gene is called a cloning vector

b) Bacterial plasmids are easy to get and change, easy to place into
bacteria and have rapid multiplication
c) Gene cloning helps to copy genes to make a protein product for medical
and research purposes.
Using Restriction Enzymes to Make a Recombinant DNA Plasmid

a) Bacterial restriction enzymes cut DNA molecules at specific DNA

sequences called restriction sites
b) A restriction enzyme usually makes many cuts, yielding restriction
fragments
Using Restriction Enzymes to Make a Recombinant DNA Plasmid

a) The most useful restriction enzymes cut DNA in a staggered way to form
‘sticky ends’ that will attach to sticky ends of other fragments.

b) DNA ligase will seal the bonds between restriction fragments

 Bacterial
extra plasmid
restriction enzymes

Restriction site
5′ 3′
G AAT T C
DNA
C T T AAG
3′ 5′
1 Restriction enzyme cuts
the sugar-phosphate dna ligase
backbones at each arrow.
3′ 5′
5′ 3′

5′ 3′
3′ 5′
Sticky end
5′
3′

3′
5′
Fragment from different DNA molecule
cut by the same restriction enzyme
5′ 3′ 5′ 3′ 5′ 3′
G AAT T C G AAT T C
C T TA A G C T TAA G
3′ 5′ 3′ 5′ 3′ 5′
One possible combination
3 DNA ligase
seals the strands.
insulin
5′ 3′

3′ Recombinant DNA molecule 5′

Recombinant
plasmid
Animation: Restriction Enzymes
a) To separate and visualize the fragments produced, gel electrophoresis
would be carried out
b) This technique uses a gel made of a polymer to separate a mixture of
nucleic acids or proteins based on size, charge, or other physical
properties
Figure 19.7
Mixture of Power
DNA mol- source
ecules of Cathode Anode
different
sizes
Wells
Gel

(a) Negatively charged DNA molecules move

toward the positive electrode.

Restriction fragments
(size standards)
(b) Shorter molecules are slowed down less than
longer ones, so they move faster through the gel.
Amplifying DNA: The Polymerase Chain Reaction (PCR) and Its Use in
DNA Cloning

a) The polymerase chain reaction, PCR, can produce many copies of a

specific target segment of DNA
b) PCR uses a special heat-stable DNA polymerase called Taq polymerase
c) PCR uses a pair of primers specific for the sequence to be amplified
 Technique 5′ 3′
Target
sequence
Genomic DNA 3′ 5′ tap polymerase
1 Denaturation 5′ 3′

3′ 5′
PCR produces a 2 Annealing
Cycle 1
chain reaction yields
Primers
2
that produces an molecules
3 Extension
exponentially New
growing nucleotides

population of
identical DNA Cycle 2
yields
molecules 4
molecules

Cycle 3 yields 8
molecules;
2 molecules
(in white boxes)
match target
sequence

Results After 30 more cycles, over 1 billion (109) molecules match the target sequence.
Animation: Cloning a Gene
 Reverse transcriptase-polymerase chain reaction DNA in
(RT-PCR) nucleus
mRNAs in
cytoplasm

1. Since eukaryotic gene expression has Reverse transcriptase

to deal with introns, scientists use mRNA Poly-A tail
5′ A A A A A A 3′
complementary DNA (cDNA), which 3′ T T T T T 5′

is complementary to the mRNA. cDNA DNA Primer

only has exons. once u ahave the dna it multiplies strand (poly-dT)
5′ A A A A A A 3′
2. The enzyme used is Reverse 3′ T T T T T 5′

transcriptase to get cDNA which can

then be used as a template for PCR
5′ 3′
amplification of the gene of interest 3′ 5′
DNA
polymerase

5′ 3′
3′ 5′
cDNA
Technique
1 cDNA synthesis mRNAs

cDNAs
Primers
2 PCR amplification

Specific
gene
3 Gel electrophoresis

Results Embryonic stages

1 2 3 4 5 6
Concept 19.2: Biologists use DNA technology to study gene expression
and function

a) Analyzing when and where a group of genes is expressed can provide

important clues about gene function
b) The easiest way to discover which genes are expressed in certain cells
is to identify the mRNAs being made
c) mRNA can be detected by nucleic acid hybridization with complementary
molecules (nucleic acid probes)
In situ hybridization uses fluorescent dyes attached to probes to identify the
location of specific mRNAs in place in the intact organism

wg mRNA en mRNA
Cells Cells
expressing expressing
the wg gene the en gene
Head Thorax Abdomen

50 µm T1 T2 T3 A1 A2 A3 A4 A5
Studying the Expression of Interacting Groups of Genes

a) Automation has allowed scientists to measure the expression of

thousands of genes at one time using DNA microarray assays
b) DNA microarray assays compare patterns of gene expression in
different tissues, at different times, or under different conditions
c) DNA microarray assays may contribute to understanding of disease and
suggest new diagnostic targets
Each dot is a well containing identical copies
of DNA fragments that carry a specific gene.
Genes expressed
in first tissue.

Genes expressed
in second tissue.

Genes expressed
in both tissues.

Genes expressed
► in neither tissue.
DNA microarray
(actual size)
DNA microarray

a) The mRNA that is obtained from the samples is colour-coded with

fluorescent tags and used to make a DNA copy (cDNA) which is then
applied to the microarray.
b) The cDNA binds to complementary base pairs in each of the spots on
the array, a process known as hybridization.
c) Based on how the DNA binds together, each spot will appear red, green,
or yellow (a combination of red and green) when scanned with a laser.
This is then interpreted by the computer software.
Determining Gene Function
what happen if u mutate the gene ?

a) One way to determine function is to disable the gene and observe the
consequences
b) Using in vitro mutagenesis, mutations are introduced into a cloned gene,
altering or destroying its function
c) When the mutated gene is returned to the cell,
the normal gene’s function might be determined by examining the mutant’s
phenotype
Determining Gene Function

how genes express their selves with the massage

a) Gene expression can also be silenced using RNA interference (RNAi)

b) Synthetic double-stranded RNA molecules matching the sequence of a
particular gene are used to break down or block the gene’s mRNA
SNPs (single nucleotide polymorphisms),

a) These are single nucleotide variants and are useful genetic markers
b) Scientists analyze the genomes of many people with a certain genetic
condition to try to find nucleotide changes specific to the condition
c) SNP variants that are found frequently associated with a particular inherited
disorder alert researchers to the most likely location for the disease-causing
gene
d) SNPs are rarely directly involved in the disease; they are most often in
noncoding regions of the genome
 A
DNA
T
Normal allele
SNP

C
G
Disease-causing
allele
Concept 19.3: Cloned organisms and stem cells are useful for basic
research and other applications

a) Organismal cloning produces one or more organisms genetically

identical to the “parent” that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce
itself indefinitely, or under certain conditions can differentiate into
one or more types of specialized cells

agriculture
Cloning Plants: Single-Cell Cultures

a) In plants, cells can dedifferentiate and then give rise to all the specialized
cell types of the organism
b) A totipotent cell is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
 The cloning of a whole carrot plant from a single carrot cell

Cross section
of carrot root

Small
fragments

Fragments Single Embryonic Plantlet was Adult plant

were cultured cells free in plant cultured on
in nutrient suspension developed agar medium.
medium; began to from a Later it was
stirring divide. cultured planted in soil.
caused single single cell.
cells to shear
off into the
liquid.
Cloning Animals: Nuclear Transplantation

a) In nuclear transplantation, the nucleus of an unfertilized egg cell or

zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown that
a transplanted nucleus can often support normal development of the egg
c) However, the older the donor nucleus, the lower the percentage of
normally developing tadpoles
Experiment Frog embryo Frog egg cell Frog tadpole
UV

Fully differ-
Less differ- entiated
entiated cell (intestinal) cell

Donor Donor
nucleus Enucleated nucleus
trans- egg cell trans-
planted Egg with donor nucleus planted
activated to begin
Results development

Most develop Most stop developing

into tadpoles. before tadpole stage.
Reproductive Cloning of Mammals

a) In 1997, Scottish researchers announced the birth of Dolly, a lamb

cloned from an adult sheep by nuclear transplantation from a
differentiated mammary cell
 Technique

Mammary Egg cell

cell donor donor

1 2
The groundbreaking Egg cell
technique used from ovary Nucleus
removed
involved transferring Cultured
mammary
3 Cells fused

the nucleus of an cells

adult cell into an

unfertilized egg cell Nucleus from
mammary cell
whose nucleus had 4 Grown in culture

been removed. Early embryo

5 Implanted in uterus
of a third sheep

Surrogate
mother
6 Embryonic
development
Lamb (“Dolly”)
Results genetically identical to
mammary cell donor
 Technique

Mammary Egg cell

cell donor donor

1 2
An electric shock Egg cell
stimulated the hybrid from ovary Nucleus
removed
cell to begin dividing Cultured
mammary
3 Cells fused

and generate an cells

embryo which was

then implanted into Nucleus from
mammary cell
the womb of a 4 Grown in culture

surrogate mother. Early embryo

5 Implanted in uterus
of a third sheep

Surrogate
mother
6 Embryonic
development
Lamb (“Dolly”)
Results genetically identical to
mammary cell donor
Reproductive Cloning of Mammals

a) Dolly’s premature death in 2003, as well as her arthritis, led to

speculation that her cells were not as healthy as those of a normal sheep,
possibly reflecting incomplete reprogramming of the original transplanted
nucleus
a) Since 1997, cloning has been demonstrated in many mammals,
including mice, cats, cows, horses, mules, pigs, and dogs
b) CC (for Carbon Copy) was the first cat cloned; however, CC differed
somewhat from her female “parent”
c) Cloned animals do not always look or behave exactly the same
Faulty Gene Regulation in Cloned Animals

a) In most nuclear transplantation studies, only a small percentage of

cloned embryos have developed normally to birth, and many cloned
animals exhibit defects
b) Epigenetic changes, such as acetylation of histones or methylation of
DNA, must be reversed in the nucleus from a donor animal in order for
genes to be expressed or repressed appropriately for early stages of
development
Stem Cells of Animals

a) Stem cells are relatively unspecialized cells that can both reproduce
indefinitely and, under certain conditions, differentiate into one or more
specialized cell types
 Stem cell derived from the bone marrow

Cell division

Stem cell and Precursor cell

Fat cells or Bone or White

cells blood cells
Embryonic and Adult Stem Cells

a) Many early embryos contain stem cells capable of giving rise to

differentiated embryonic cells of any type
b) In culture, these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions, they can be made to differentiate into a
variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are
used in the body to replace nonreproducing cells as needed
 Embryonic Adult stem
Researchers found stem
stem cells cells, cells in the bone marrow,
in this example bone marrow skin, hair, eyes.

Cells that can generate Cells that generate a limited

all embryonic cell types number of cell types

Cultured stem cells

Different culture
conditions

Liver cells Nerve cells Blood cells

Different types of
differentiated cells
 ethical issues

a) Embryonic stem (ES) cells are pluripotent, capable of differentiating into

many different cell types
b) The ultimate aim of research with stem cells
is to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
Induced Pluripotent Stem (iPS) Cells

a) Researchers can treat differentiated cells, and reprogram them to act like
ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell
master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions of
ES cells
d) iPS cells can be used as models for study of certain diseases and
potentially as replacement cells for patients
Concept 19.4: The practical applications of DNA-based biotechnology
affect our lives in many ways

a) Many fields benefit from DNA technology and genetic engineering

b) Medical applications include identifying genes involved in genetic
diseases.
c) Gene therapy
Human Gene Therapy

a) Gene therapy is the alteration of an afflicted individual’s genes

b) Gene therapy holds great potential for treating disorders traceable to a
single defective gene
c) Vectors are used for delivery of genes into specific types of cells, for
example bone marrow
d) Gene therapy provokes both technical and ethical questions
 Cloned
gene 1 Insert RNA version of normal allele
into retrovirus or other viral vector.

Viral RNA

2 Let virus infect bone marrow cells

Viral that have been removed from the
capsid patient and cultured.

basic
3 Viral DNA carrying the normal
allele inserts into chromosome.

Bone
marrow
cell from
patient

4 Inject engineered Bone

cells into patient. marrow
Pharmaceutical Products

a) Advances in DNA technology and genetic research are important to the

development of new drugs to treat diseases
b) Synthesis of Small Molecules for Use as Drugs
c) The drug imatinib is a small molecule that inhibits overexpression of a
specific leukemia-causing receptor
d) This approach is feasible for treatment of cancers in which the molecular
basis is well-understood
Protein Production in Cell Cultures

a) Host cells in culture can be engineered to secrete a protein as it is made,

simplifying the task of purifying it
b) This is useful for the production of insulin, human growth hormones, and
vaccines
Protein Production by “Pharm” Animals

a) Transgenic animals are made by introducing genes from one species

into the genome of another animal
b) Transgenic animals are pharmaceutical “factories,” producers of large
amounts of rare substances for medical use
Forensic Evidence and Genetic Profiles

a) An individual’s unique DNA sequence, or genetic profile, can be

obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called
short tandem repeats (STRs). These are tandemly repeated units of 2
to 5 nucleotides sequences in specific region of the genome. The
number of repeats present in these regions is highly variable from
person to person
(a) Earl Washington just
before his release in 2001,
after 17 years in prison.

Source of STR STR STR

sample marker 1 marker 2 marker 3
DNA on victim 17,19 13,16 12,12

Earl Washington 16,18 14,15 11,12

Kenneth Tinsley 17,19 13,16 12,12

(b) This data led Tinsley to plead guilty to the crime.

Agricultural Applications

a) DNA technology is being used to improve agricultural productivity and

food quality
b) The Ti plasmid is the most commonly used vector for introducing new
genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful
genes including those for herbicide resistance, increased resistance to
pests, increased resistance to salinity, and improved nutritional value of
crops
Video: Pronuclear Injection