CHAPTER 20: DNA TOOLS AND BIOTECHNOLOGY
Concept 20.1. DNA Sequencing and DNA cloning are
valuable tools for genetic engineering and biological
inquiry
Nucleic Acid Hybridization- the base pairing of one
strand of a nucleic acid to a complementary sequence
on a strand from a different nucleic acid molecule.
A. DNA Sequencing- process to determine the complete
nucleotide sequence of a DNA molecule
Steps in DNA Sequencing
1. DNA is first cut into fragments
2. Each fragment is sequenced
Dideoxyribonucleotide (or dideoxy) Chain Termination
Sequencing
- Was used by the first automated procedure
- one strand of a DNA fragment is used as a
template for synthesis of a nested set of
complementary fragments; these are further
analyzed to yield the sequence
- Biochemist Frederick Sanger received the Nobel
Prize in 1980 for developing this method
- is still used for routine small-scale sequencing
jobs.
- II. the plasmid is returned to bacterial cell producing
Sequencing by Synthesis
- specific strand of each fragment is immobilized,
and the complementary strand is synthesized,
one nucleotide at a time.
- enables electronic monitors to identify in real
time which of the four nucleotides is added
- each about 300 nucleotides long, are sequenced
in parallel in machines, accounting for the high
rate of nucleotides sequenced per hour.
B. Making Multiple Copies of a Gene or other DNA
Segment
DNA Cloning - methods for preparing well-defined
segments of DNA in multiple identical copies
Plasmid
- small, circular DNA molecules that are
replicated separately
- found in bacteria
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Recombinant DNA molecule
- a molecule containing DNA from two different
sources, very often different species
Cloning Vector
- a DNA molecule that can carry foreign DNA into
a host cell and be replicated there
Gene Cloning
- production of multiple copies of a single gene is
a type of DNA cloning
Why is Gene Cloning used?
a. to make many copies of, or amplify, a
particular gene
b. to produce a protein product from it
Genetically Modified Organism
- a resistance gene present in one crop species
might be cloned and transferred into plants of
another species
Gene Cloning in Bacteria:
I. Isolated plasmid of a bacterium will be inserted with a
DNA form another “source” resulting to recombinant
DNA molecule
recombinant bacterium
Recombinant bacterium
- reproduces through repeated cell divisions to
form a clone of cells, a population of genetically
identical cells
III. Dividing bacteria replicate the recombinant plasmid
and pass it on to their descendants, the foreign DNA
and any genes it carries are cloned at the same time.
Why are bacterial plasmids are used as cloning
vectors?
a. Readily obtained from commercial suppliers
b. easy to manipulate to form recombinant plasmids
c. easy to re-introduce to the cell.
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C. Using Restriction Enzymes to make a recombinant
DNA Plasmid
Restriction Enzymes
- were discovered in the late 1960s by biologists
doing basic research on bacteria
- protect the bacterial cell by cutting up foreign
DNA from other organisms or phages
- enzymes that cut DNA molecules at a limited
number of specific locations.
Restriction Site
- a particular short DNA sequence where
restriction will start
- mostly symmetrical when sequence of
nucleotides is same on both strands when read
in 5” -3”
Restriction Fragments
- cuts in such a DNA molecule
- “All copies of a given DNA molecule always yield
the same set of restriction fragments when
exposed to the same restriction enzyme.”
Sticky End
- one single-stranded end
- These short extensions can form hydrogen-
bonded base pairs with complementary sticky
ends on any other DNA molecules cut with the
same enzyme
Gel Electrophoresis
- uses a gel made of a polymer as a molecular
sieve to separate out a mixture of nucleic acid
fragments by length
- used in conjunction with many different
techniques in molecular biology.
D. Amplifying DNA: PCR and its use in DNA Cloning
Polymerase Chain Reaction (PCR)
- devised in 1985
Steps in PCR:
1. Denaturation – increase temperature to 94 – 96
degrees Celsius
 Breaking bonds
 Unzipping double stranded DNA
 Breaks H bond
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2. Annealing – decrease temperature to -68 degrees
Celsius
 To stick something on something, to put them
together
 You put primer on denatured DNA strand
 Primer – starts the elongation of
complementary DNA Strand eventually
replicating DNA, it is a short segment of DNA
 Primer sticks on very short sequences of bases
(50 – 100 bases), hahanapin ang
complement/counterpart
3. Elongation – increase temperature to 72 degrees
Celsius
 New DNA strand elongates
 Complementary strands elongate (started by
primer)
 Bases/building blocks will be added to primer
 Oligonucleotides (DNTPs) – separate
nitrogenous bases
 DNA polymerase gets the nitrogenous bases
and sticks it to the primer, stitches building
blocks (Thermophilus aquaticus -Taq
polymerase) – microbe that lives in high
temperature
 5’ – 3’ direction of elongation
 Pfu polymerase from Pyrococcus furiosus can
also be used but is more expensive than Taq
E. Expressing Cloned Eukaryotic Genes
a. Bacterial Expression Systems
Expression Vector
- a cloning vector that contains a highly active
bacterial promoter just upstream of a
restriction site where the eukaryotic gene can
be inserted in the correct reading frame.
Disadvantages:
- Presence of noncoding regions (introns) in
most eukaryotic genes. Introns make gene very
long thus, it prevents correct expression by
bacterial cells. This can be solved with exons-
only gene.
CHAPTER 20: DNA TOOLS AND BIOTECHNOLOGY
b. Eukaryotic DNA Cloning and Expression Systems
(EDCES)
- yeast can be used for cloning since they are
easy to grow and have plasmids
Advantage:
a. Many eukaryotic proteins will not function
unless they are modified after translation.
Bacterial cells cannot modify the proteins and
some genes cannot be modified even by yeast
(addition of carbohydrates- glycosylation) thus,
cultured cell types or lines.
Electroporation - a brief electrical pulse applied to a
solution containing cells creates temporary holes in
their plasma membranes, through which DNA can
enter.
Other techniques for EDCES:
a. Inject DNA directly into single eukaryotic cells using
microscopically thin needles
b. Using the soil bacterium Agrobacterium tumefaciens
F. Cross-Species Gene expression and Evolutionary
Ancestry
Pax-6 Gene
- triggers a complex program of gene expression
resulting in formation of the vertebrate eye,
-which has a single lens
- it has different effect when placed in different
culture
Concept 20.2. Biologists use DNA technology to study
gene expression and function
A. Analyzing Gene Expression
a. Studying Expression of Single Genes
Nucleic Acid Probe
- The complementary molecule, a short, single-
stranded nucleic acid that can be either RNA or
DNA
- Using our cloned gene as a template, we can
synthesize a probe complementary to the
mRNA.
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mRNA detection Techniques:
a. reverse Transcriptase Polymerase Chain or
RT-PCR
- starts turning sample sets of mRNAs
into double-stranded DNAs with the
corresponding sequences
Steps in RT-CPR:
1. enzyme reverse transcriptase (from a retrovirus ) is
used to synthesize a complementary DNA copy of each
mRNA in the sample (reverse transcript)
2. a short complementary strand of thymine
deoxyribonucleotides (poly-dT) to be added and used as
a primer for synthesis of this DNA strand
3. Following enzymatic degradation of the mRNA, a
second DNA strand, complementary to the first, is
synthesized by DNA polymerase.
4. quantitative RT-PCR (qRT-PCR) uses a fluorescent dye
that fluoresces only when bound to a doublestranded
PCR product
b. In situ hybridization
- Each probe molecule is labeled during
synthesis with a fluorescent tag so we can
follow it. A solution containing probe molecules
is applied, allowing the probe to hybridize
specifically with any complementary sequences
on the many mRNAs in cells in which the gene is
being transcribed.
Complementary DNA (cDNA) – resulting double
stranded DNA
b. Studying the Expression of Interacting
Groups of Genes
Systems approach
- study the expression of large groups of genes
DNA microarray assays
- consists of tiny amounts of a large number of
single-stranded DNA fragments representing
different genes fixed to a glass slide in a tightly
spaced array, or grid, of dots.
- used to identify sets of genes co-expressed by a
group of cells
CHAPTER 20: DNA TOOLS AND BIOTECHNOLOGY
RNA sequencing
- simply sequence the cDNA samples from
different tissues or different embryonic stages
in order to discover which genes are expressed
B. Determining Gene Function
a. Editing Genes and Genomes
In vitro Mutagenesis
- specific mutations are introduced into a cloned
gene, and the mutated gene is returned to a cell
in such a way that it disables (“knocks out”) the
normal cellular copies of the same gene.
CRISPR-Cas9 system
- a powerful new technique for gene editing in
living cells and organisms.
- used to identify sets of genes co-expressed by a
group of cells
Gene drive
- engineering the new allele so that it is much
more highly favored for inheritance than is the
wild-type allele
- variety of specialized cells
Other methods for Studying Gene Function:
a. Genome-wide association studies
- analyze the genomes of large numbers of
people with a certain phenotypic condition or disease,
such as heart disease or diabetes, to try to find
differences they all share compared with people
without that condition
b. Single nucleotide polymorphism (SNP)
- A single base-pair site where variation is found
in at least 1% of the population
Concept 20.3. Cloned Organism and Stem Cells use for
research and other applications
Stem cell
- unspecialized cell that can both reproduce itself
indefinitely and, under appropriate conditions,
differentiate into specialized cells of one or
more types. Stem cells have great potential for
regenerating damaged tissues
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A. Cloning Plants: Single- Cell Cultures
Totipotent
- mature cells can “dedifferentiate” and then
give rise to all the specialized cell types of the
organism
B. Cloning Animals: Nuclear Transplantation
Somatic cell nuclear transfer (nuclear transplantation)
- remove the nucleus of an egg (creating an
enucleated egg) and replace it with the nucleus
of a differentiated cell, a procedure called
nuclear transplantation
a. Reproductive Cloning of Mammals
o production of new individuals
C. Stem Cells of Animals
Stem Cells
- can be isolated from early embryos at a stage
called the blastula stage
a. Embryonic Stem (ES) Cells
o reproduce indefinitely
o can be made to differentiate into a
Adult Stem Cells
o adult stem cells are not able to give rise
to all cell types in
pluripotent
- capable of differentiating into many different
cell types.
Therapeutic Cloning
- main aim of cloning is to produce ES cells to
treat disease
b. Induced Pluripotent Stem (iPs) Cells
o researchers transformed the
differentiated cells into a type of ES cell
by using a retrovirus to introduce extra,
cloned copies of four “stem cell” master
regulatory genes
o perform most of the functions of ES
cells, but there are some differences in
gene expression and other cellular
functions, such as cell division.
CHAPTER 20: DNA TOOLS AND BIOTECHNOLOGY
Major Uses of iPs Cells:
a. Act as model cells for studying the disease and
potential treatments
- cells from patients suffering from diseases
have been reprogrammed to become iPS cells
b. In the field of regenerative medicine, a patient’s own
cells could be reprogrammed into iPS cells and then
used to replace nonfunctional tissues, such as insulin-
producing cells of the pancreas
20.4. Practical Applications of DNA-based technology
affect our lives in many ways
Medical Applications
- identification of human genes whose mutation plays a
role in genetic diseases
- contributing to our understanding of “nongenetic”
diseases
- DNA microarray assays or other techniques to
compare gene expression in healthy and diseased
tissues, researchers are finding genes that are turned on
or off in particular diseases. These genes and their
products are potential targets for prevention or
therapy.
A. Diagnoses and Treatment of Diseases
- use of PCR and labeled nucleic acid probes to
track down pathogens
- RT-PCR is often the best way to detect an
otherwise elusive infective agent.
- genome-wide association studies have
pinpointed SNPs (single nucleotide
polymorphisms) that are linked to disease-
associated alleles
- prompted improvements in disease
treatments
B. Human Gene Therapy and Gene Editing
Gene therapy
- the introduction of genes into an afflicted
individual for therapeutic purposes
- The aim of this approach is to insert a normal
allele of the defective gene into the somatic
cells of the tissue affected by the disorder.
- Bone marrow cells, which include the stem cells
that give rise to all the cells of the blood and
immune system, are prime candidates.
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CRISPR-Cas9
C. Pharmaceutical Products
- development of useful drugs to treat diseases
- Pharmaceutical products are synthesized using
methods of either organic chemistry or
biotechnology
D. Synthesis of Small Molecules for Use as Drugs
imatinib (trade name Gleevec)
- small molecule that inhibits one tyrosine kinase.
The overexpression of this kinase, resulting
from a chromosomal translocation, is
instrumental in causing chronic myelogenous
leukemia
E. Protein Production in Cell Cultures
- Pharmaceutical products that are proteins are
commonly synthesized on a large scale using
cell cultures
- ex. human insulin and human growth
hormone
- tissue plasminogen activator (TPA). If
administered shortly after a heart attack, TPA
helps dissolve blood clots and reduces the risk
of subsequent heart attacks.
F. Protein Production by “Pharm” Animals
Introduce a gene (or other DNA) from an animal
into the genome of another individual, often of a
different species. This individual is then called a
transgenic animal.
Transgene – foreign DNA
G. Forensic Evidence and Genetic Profiles
- body fluids or small pieces of tissue may be
left at the scene or on the clothes or other
possessions of the victim or assailant
DNA testing - can identify the guilty individual with a
high degree of certainty because the DNA sequence of
every person is unique (except for identical twins)
Genetic profile (dna fingerprint) - individual’s unique
set of genetic markers
Short tandem repeats (STRs) - tandemly repeated units
of two- to five-nucleotide sequences in specific regions
CHAPTER 20: DNA TOOLS AND BIOTECHNOLOGY

of the genome. The number of repeats present in these

regions is highly variable from person to person
(polymorphic), and even for a single individual, the two
alleles of an STR may differ from each other

H. Environmental Cleanup
- diverse abilities of certain microorganisms to
transform chemicals are being exploited for
environmental cleanup
- Genetically engineered microorganisms may
become important in both mining (especially as
ore reserves are depleted) and cleaning up
highly toxic mining wastes.
- Biotechnologists are also trying to engineer
microorganisms that can degrade chlorinated
hydrocarbons and other harmful compounds
- These microorganisms could be used in
wastewater treatment plants or by
manufacturers before the compounds are ever
released into the environment.

I. Agricultural Applications
- DNA technology enables scientists to produce
transgenic animals, which speeds up the
selective breeding process.
- Agricultural scientists have already endowed a
number of crop plants with genes for desirable
traits
- Crops engineered with a bacterial gene making
the plants resistant to an herbicide can grow
while weeds are destroyed, and genetically
engineered crops that can resist destructive
insects reduce the need for chemical
insecticides.

J. Safety and Ethical Questions Raised by DNA

Technology
- One safety measure is a set of strict laboratory
procedures designed to prevent engineered
microorganisms from infecting researchers or
accidentally leaving the laboratory

Genetically modified organisms (GMOs)

- a transgenic organism, one that has acquired by
artificial means one or more genes from
another species or even from another variety of
the same species

Chapter 20. DNA TOOLS AND BIOTECHNOLOGY | ALQUERO