DNA Technology

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 DNA Technology
Biotechnology - use of organisms to
perform specific manufacturing
processes

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 DNA Identification
Except identical twins, no two people
in the world are genetically the same
From person to person, the entire
human genome varies about 0.10%
Because of that 0.10% difference,
scientists can identify every individual
based on DNA 3
DNA ID compares samples in regions
of chromosome that are different
Useful for many purposes, including
determining paternity (who’s the baby
daddy), identifying human remains
(dead bodies/skeletons), tracing
human origins, and providing
evidence in criminal cases (CSI!!!!)
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 Noncoding DNA
About 98% of our DNA does NOT
code for any protein
Noncoding DNA
Contains length polymorphisms –
variations in length of DNA molecule
between known genes
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Some LPMs in noncoding regions
come from short, repeating sequences
of DNA
Ex. Sequence might be CACACA…..

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These can repeat a few or many times in
tandem (one “behind” another – like a
tandem bicycle)
Variable number tandem repeats (VNTR)
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Number of tandem repeats varies
among individuals
For each VNTR in DNA, person will
have certain number of repeats
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Geneticists calculate how frequently VNTR
used in DNA ID happen in general
population
That way they can determine how rare a
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 Steps in DNA ID
Isolate DNA in sample and make
copies
Cut DNA into shorter pieces that
contain known VNTR areas
Sort DNA by size

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Compare size fragments in unknown sample
to those of known samples
If match occurs, person’s ID confirmed
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 Copying DNA: Polymerase
Chain Reaction

Often DNA recovered from crime

scene or human tissue is in small
amounts
Scientists need to make copies to
have enough for DNA ID 13
Polymerase chain
reaction (PCR) is
technique that
quickly produces
many copies of
DNA fragment

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PCR requires a template
– DNA fragment
containing sequence to
copy
Also requires supply of 4
nucleotides, heat-tolerant
DNA polymerase, and
primers

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Primers – artificially made pieces of single-
stranded DNA 20-30 nucleotides long
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Primers must be present for
DNA polymerase to begin
replication
Complementary to ends of DNA
pieces to be copied

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When everything combined, original
DNA is heated to denature the molecule
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copying
begins
Primers bind
to DNA

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DNA polymerase makes copy of both
strands
Heating breaks bonds holding template DNA
to newly made strands 20
After cooling, primers bind to DNA
again
Cycle repeats – DNA polymerase
copies both strands, heating breaks
bonds, cooling, primers bind again….
With every cycle DNA doubles

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 Cutting DNA:
Restriction Enzymes
Genetic engineers use
restriction enzymes to
cut DNA into
manageable pieces
Each recognizes specific
sequence of DNA
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Created by restriction Sticky Ends
enzymes cutting out piece of
DNA
Sticky end - single-stranded
piece of DNA that readily
binds to complementary
chains of DNA
Same restriction enzymes
used – sticky ends can bind
together 24
Gel Electrophoresis
Separates fragments
based on size and
charge

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DNA samples cut
with restriction
enzyme
Places in wells
made on thick gel

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Electric current run
through gel
(-) charged
fragments migrate to
positive end of gel
Smaller pieces
migrate faster, larger
pieces slower
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DNA transferred to nylon membrane
Radioactive nucleic acid probes added
Probes bind to complementary DNA

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X-ray film exposed to radio-labeled
membrane
Resulting pattern of bands is DNA
fingerprint

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 Accuracy of DNA Fingerprints
Specific sections of DNA compared
Every human DNA has noncoding
repeated sequences
Scientists compare 5 different sites
between people
Chance that 2 people match all 5 sites is
less 1 in 1 million 32
 Recombinant DNA
Techniques of DNA technology used to
modify genome of living cell or
organism
Genetic engineering
Recombinant DNA results when DNA
from two different organisms is joined
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 Example of
Recombinant DNA
To study blood vessel growth, researchers
combined jellyfish gene encoding green
fluorescent protein (GFP) that glows under
UV light with zebrafish gene involved in
blood-vessel development
They inserted GFP/blood-vessel gene into
zebrafish embryos 34
Fish’s blood-vessel cells
transcribed recombinant DNA
Made green fluorescent
proteins
As zebrafish grew their blood
vessels glowed green
More easily studied
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 Cloning Vector
Clone ☞ exact copy of DNA segment,
whole cell, complete organism
Researchers clone DNA pieces by putting
them in vectors ☞ DNAs that can
replicate within a cell and that can carry
foreign DNA
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When vector carrying foreign DNA
enters bacteria and they reproduce,
they grow colony of cloned cells that
includes foreign DNA

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Plasmids ☞ rings of DNA found
naturally in some bacterial cells in
addition to main chromosome 39
Plasmid and DNA of interest (insulin in
this case) are isolated
Restriction enzyme is used to cut DNA
into many fragments 40
Sticky ends hold donor and plasmid
DNA together
Enzyme called DNA ligase joins them
permanently 41
 Donor Gene and Gene
Clone
Donor gene - gene isolated from another
organism
Spliced into plasmid
When bacteria reproduces, plasmid
reproduces donor gene (gene clone)

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 Recombinant plasmids transferred
into bacteria
When bacteria copy their own DNA
they also copy plasmid with donor
gene
After cells grow into colonies,
bacterial colony with plasmid with
desired DNA ID’ed by using a probe
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 Probes
Probe ➽ strand of RNA or single-
stranded DNA that is labeled with
radioactive element or fluorescent dye
Can base pair to specific DNA - like
donor gene in recombinant DNA
For insulin, probe is mRNA
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To see which of the 1000s of colonies has
recombinant gene, DNA transferred from
bacteria to filter paper
When viewed under UV light or exposed to
photographic film, clone of cells with donor
DNA and probe glows
Biologists can now grow more of the clone
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 Applications for DNA
Technology
Identify criminals
Free people innocent but were
convicted

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Anthropologists use DNA ID to trace
human origins and migrations
Environmentalists use to trace threatened
and endangered species
Give microorganisms new capabilities
Mass production of insulin for diabetes
patients
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Since 1982, more than 30 products made
using DNA technology
Factors to treat immune system
deficiencies and anemia
Clotting factors for people with hemophilia

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Human growth hormone for people with
growth defects
Interferons for viral infections and cancer
Growth factors to treat burns and ulcers
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One exciting application of DNA technology has been
the sequencing of the entire human genome. This
section discusses how researchers used modern
genetic tools to sequence the human genome and
what their findings mean for 21st century biology and
society.

THE HUMAN GENOME

PROJECT

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 Mapping the Human
Genome

In 1990 geneticists around the world

started the Human Genome Project
Research effort to sequence all of our DNA
and locate all the important sequences

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Seeks to determine the sequence of all
3.3 billion nucleotides
Map location of every gene on each
chromosome
Will provide insight to our evolutionary
past, genome organization, gene
expression, cell growth, and health
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More than 20 labs in 6 different
countries took part in project
Sequence completed in 2003 (two
years ahead of schedule)

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 Important Understandings

Only 2% of the human genome codes for

proteins
Chromosomes have unequal distribution
of exons
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Genome smaller than estimated
20-25,000 protein-encoding genes
Previously thought to be 100,000
Many RNAs not only used for translation but
also regulating gene expression

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Exons of human genes spliced in
many ways - allows same gene to
encode different versions of a protein
Proteome - complete set of proteins
Human proteome very complex

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About ½ of the genome comes from
shuffling transposons - pieces of DNA that
move from one chromosome location to
another
Transposons appear to have no specific
role in development or physiology

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Genome has about 8 million single
nucleotide polymorphisms (SNP)
These are unique spots where
individuals differ by a single nucleotide
Important for mapping genome in
more detail
Important in ID’ing human disease
genes 58
 Model Species
To better understand how human genes
control development and health and
explain how genes affect behavior
biologists want to map similar genes in
model species
Since HGP, many other genome sequence
projects have been completed 59
Researchers can cause mutations in
different species and look for gene
actions
Represent broad base of organisms –
possible to make generalizations that
relate to larger groups
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 Applications
Scientists already discovered specific
genes responsible for genetic
disorders
May improve diagnoses, treatments,
therapies

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 The Future of
Genomics
Although more than 3 billion letters of genetic code
have been sequenced, it is only the beginning
New tools and fields of research now enable
acquisition, analysis, storage, modeling, distribution
of information
Geneticists can now apply new tools to benefit
human health
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 Bioinformatics

Combines biological science,

computer science, information
technology
Uses databases to store and mix data
coming out of genomic research
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One database called BLAST allows
fast comparisons between gene or
protein sequence from one organism
and similar sequences from all other
organisms in database at National
Center for Biotechnology Information
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BLAST allows a worker to find
matching genes in different organisms
By knowing function of gene from
related model species (mouse),
researcher can infer function of human
gene
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 Proteomics

Study of all of an organism’s proteins

including identity, structure, interaction,
abundance

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Key tool is two dimensional gel
electrophoresis - separates protein into
individual spots
Researcher can cut protein spot from gel
Use special methods to determine amino
acid sequence
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Using bioinformatics, search DNA of
sequenced genome and match individual
gene to that protein
Proteomics and bioinformatics allow
medical researchers to ID new targets for
theraputic drugs and develop new
markers (criteria) for diagnosing disease
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 Microarrays
Two dimensional arrangement of DNA
molecules representing thousands of
cloned genes
Can show which genes are active in cell
Robotic machines arrange tiny amounts of
sequences from thousands of genes on
microscope slide 69
 Ex. How tumor cells differ from
normal cells
mRNA from tumor labeled with
fluorescent dye and poured onto
microarray slides
More mRNA that binds to complement
on particular spot on slide – more color
Indicates that gene is highly active
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Doctors use microarrays to classify
patients’ cancers
Can lead to better-informed decision
about what type of treatment is best

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In addition to DNA fingerprints and genomics,
genetic engineering techniques are being used
in medical, industrial, commercial, and
agriculture settings. This section discusses
some of these applications and the ethical
issues the techniques raise.
GENETIC ENGINEERING

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 Medical Applications

Genetic engineering allows biologists to

study how genes function
Ex. In Montreal (Canada) they used genetic
eng. To study brain development in mice
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Wanted to see what activates gene Hoxd4
as hindbrain develops in embryo
Important b/c abnormal hindbrain can
contribute to autism
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Researchers combined Hoxd4 gene and
area next to the gene with a “reporter gene”
Reporter gene codes for enzyme that
makes blue-colored product
Inserted recombinant DNA into mouse cells
Found region next to Hoxd4 could turn on
reporter gene and blue product
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When they mutated adjacent area
there was no blue color in hindbrain
There was blue color in spinal cord
Conclusion: DNA sequence next to
Hoxd4 helps control hindbrain
development

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 Gene Therapy
Treating genetic disorders by
introducing a gene into patient’s cells
Works best for disorders resulting from
loss of single protein
Ex. Cystic fibrosis

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 Cystic fibrosis
Results from lack of functional gene CFTR
When normal, gene encodes protein that
helps transport ions into and out of cells in
airways
Without gene, poor ion exchange causes
symptoms of CF
Ex. Buildup of sticky mucus that blocks
airways 78
Researchers isolate functional gene
Insert healthy gene into viral vector
Give recombinant virus to patient by
using nasal spray 79
Healthy copy of CFTR gene
temporarily makes missing protein and
improves ion exchange
traditional treatment involves thumping
sessions

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CF research has accelerated since
discovery of CFTR gene in 1989
Researchers able to add healthy copy
of CFTR into DNA of CF cells
Result – immediate return to normal
ion transport mechanism
BUT trials in lab are different than trials
on humans 81
Cells that express highest levels of
CFTR are deeper in lungs than gene
therapy can reach
b/c cells that line airway slough off
periodically treatment must be
repeated
Patients can also suffer immune
reactions to treatment 82
 Cloning

1990s – biologists started cloning whole

sheep and mice
Cloning by nuclear transfer – introducing
nucleus from a body cell into an egg cell to
make organism identical to nucleus donor
First successful cloned animal - Dolly the
sheep in 1996 83
 Scientists in Scotland isolated mature,
functioning mammary cell from adult sheep
Isolated egg cell from second sheep and
removed nucleus

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Fused mammary cell with empty egg cell
Egg stimulated to divide and grew into
embryo
Embryo planted in uterus of surrogate
mother
Dolly was born – genetically identical to
donor of mammary cell
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Dolly suffered premature aging and
disease
Died at age 6, half a normal sheep’s
lifetime

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It was found that Dolly had short telomeres -
repeated DNA sequences at ends of
chromosomes that shorten with each round
of cell division
Associated with premature aging
Other clones species have not had telomere
shortening
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Goal of animal cloning - alter the genome
in some useful way
Ex. Altered and cloned goats secrete
human blood clotting factors into their milk
Ex. Cloned pigs altered for possible organ
transplant in humans

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 Vaccines
Substance containing all or part of
harmless version of pathogen
Doctors put in body to produce immunity
to disease
Immune system recognizes surface
proteins on pathogen and responds by
making antibodies 89
 DNA Vaccine
Vaccine made by using one or more genes from
pathogen
Does not have disease-causing capability
Injected into patient
Directs synthesis of protein
Immune system defends against protein
If vaccinated person gets infected by disease the
immunity should protect them
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 Agricultural
Applications
Plant researchers using genetic engineering
to make genetically modified (GM) crops
Biologists make plants more tolerant to
environmental conditions
Also added genes to wheat, cotton,
soybeans that make it resistant to herbicides
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Transferred genes harmful to insects and
other pests to increase amount of food
produced by one crop
Plants protected from damage – yield more
food 92
Also improve nutritional value of crops
Rice has low levels of iron and beta
carotene
Needed to make vitamin A
Genetic engineers have added genes
to rice to fix the deficiency
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 Ethical Issues
Bioethics – study of ethical issues related
to DNA tech
Want to make sure none of the tools are
dangerous or yield unwanted results
Technology and data should be carefully
used
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Some people concerned that GM food
crops might harm the environment
What would happen if genes for
herbicide jumped to weeds?
mid-1970s government agencies set
standards for safety procedures
Required permits and labels for GM
products
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Most scientists agree gene therapy
unethical if it involves reproductive
cells that would affect future
generations
Ex. Cloning human embryos

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