On the following slides,
the yellow sections
identify the main points;
Underlined words =
vocabulary! Hyperlinks
are in orange.
Preparation for DNA sequencing
Maggie Bartlett, NHGRI
UNIT 9 - BIOTECHNOLOGY
Biotechnological advances in recent years has led to an
increase in our understanding of genes and improve our
ability to move and manipulate genetic information in an
organism. High tech equipment allows researchers to do
amazing things! This unit will discuss biotechnologies such
as gene splicing, cloning and review equipment and careers
relating to this growing field of medical science.
INTRODUCTION
NHGRI researcher using a pipette to load DNA into a gel.
Maggie Bartlett, NHGRI
DNA Fingerprinting
 Used in law enforcement (and as seen on CSI),
DNA fingerprinting compares the separation of
segments of DNA to find a match.
 The DNA is cut into
fragments by enzymes and
separated by a process
called gel electrophoresis to
match individuals (to a
crime or family member).
 The patterns, like
fingerprints, are individual
and unique.
 Animation
http://www.teacherthomas.com/wp-content/uploads/2010/03/GelElectrophoresis.gif
Genetic Engineering
 Scientists have the can now cut ‘n
paste genes in DNA.

EX When a gene of a firefly is inserted into
a tobacco plant, the tobacco plant will
produce the proteins to glow.
 Genetic engineering is the ability
cut (or cleave) DNA from one
organism and insert in to the DNA
of another organism (of the same
species or a different species).
http://upload.wikimedia.org/wikipedia/en/8/80/Glowin
g_tobacco_plant.jpg
What is Recombinant DNA?
 Recombinant DNA is made
by recombining fragments of
DNA from different sources.
 Such technology creates
organisms that contain DNA
from another organism,
creating a transgenic
organism.
 [AKA genetically modified
organism, GMO]
 EX modified tobacco plant
"Genetically modified organisms
(GMO)." Biotechnology: Changing
Life Through Science. Detroit:
U*X*L, 2010. Science in Context.
Web. 22 Jan. 2014.
How do Restriction Enzymes
Work?
 Restriction enzymes are bacterial
enzymes that have the ability to
cut both strands of the DNA
molecule at a specific nucleotide
sequence.
 Typically cut @ palindromic
sequences (EX AATT and TTAA)
 (Psst: palindrome- reads the same
forwards as backwards; “race car”)
http://www.ptbeach.com/cms/lib02/NJ01000839/Centricity/Do
main/113/en.gif
Restriction Enzymes Cont.
http://snhs-plin.barry.edu/cell-biologylab/Restriction_Digest_MIT_Lippert_files/100000000000052F000003E8CBEA8CE5.jpg
 If the same enzyme is
used to cleave DNA
from BOTH organisms,
the two pieces of DNA
will have matching
sticky ends and will join
together at these ends
[they will join together
again based on the
GCAT rule].
 Animation
What are Vectors?
 A vector is the means by which DNA from another
species can be carried into the host cell.
 Vectors can be biological [EX Virus or plasmid] or
mechanical [EX micropipette shown below].
IVF treatment sperm being
injected into an egg.
Photograph: Getty
Images/Science Photo Library RF
Vectors Cont.
 Plasmids are small rings of DNA found in
prokaryotes are used as vectors to carry a specific
gene of interest.
 Video
http://upload.wikimedia.org/wikipedia/commons/thumb/c/cf/Plasmid_(english).svg/300px-Plasmid_(english).svg.png
How do you Produce a Transgenic
Organism?
#1
• Isolate the foreign gene of interest using
restriction enzymes to cut DNA at specific points
#2
• Attach the DNA fragment to a carrier (AKA vector)
[A vector, such as a virus or needle, is used to
move the genes]
http://fhs-bio-wiki.pbworks.com/f/1267556976/plantplasmidtechn.jpg
#3
• Transfer it
to a host
organism
Now recombinant DNA is
used to create a variety of
end products, from
genetically engineered
plants to drugs to
specialized bacteria.
Illustration by GGS Inc.
"Recombinant DNA
Technology." Biotechnology: Changing
Life Through Science. Vol. 2: Agriculture.
Detroit: U*X*L, 2007. 468-472. Gale
Virtual Reference Library. Web. 9 Jan.
2013.
What is
PCR?
http://www.genomics.agilent.com/files/Media/8800_withHelix.jpg
 Using Polymerase Chain Reaction
machine (PCR) – lab technicians can
make millions of copies of DNA
outside of living organisms.
 Heat is used to separate the DNA
and an enzyme (DNA polymerase)
replicates the DNA [just as it would
inside the nucleus].
 The both genetic material and appropriate
enzymes are added to a PCR machine.
 Millions of copies [clones] are made
in less than a day.
 Video
What is a Clone?
 A clone is an exact genetic
copy of an organism.
 In 1958: cloned carrot.
 In 1964: cloned frog tadpole.
 In 1997, scientists cloned the first
mammal, Dolly the sheep.
 Since then, they have successfully
cloned other mammals [goats, mice,
cattle, pigs, a horse].
Dr. Hinrichs, a veterinarian and
professor at Texas A&M University
and Eric Palmer, Chairman of
Cryozootech posing with Paris
Texas, the first cloned horse in the
United States from adult horse skin
cells.
"Cloned Horse in United States." Gale Opposing Viewpoints in Context. Detroit:
Gale, 2010. Gale Opposing Viewpoints In Context. Web. 11 Jan. 2013.
Cloning & Somatic Cell
Nuclear Transfer
 In somatic cell nuclear transfer, the genetic material
from a donor cell is inserted into a nucleus-free egg.
 The egg is stimulated to initiate cell division and then
transferred in to a surrogate to “clone” the organism.
 With Dolly, genetic material was introduced into 277 eggs and only 29
cells remained alive.
 In 2008: San Diego-based Stemagen announced the formation of
embryonic cells after the transfer of nuclei from human skin cells into
“empty” human eggs.
 Animation
http://www.prolifelouisiana.org/uploads/images/Nuclear-Transfer.jpg
Human Cloning
 Cloning organisms, while
controversial, but is not
as controversial as the
thought of human
cloning.
 There are some who
argue that cloning is
“unnatural”, while others
cite cloning as a useful
tool for medicine.
 Human Cloning Article
The first cloned cat, named "cc," (short
for "copycat") is proudly displayed by
doctors Mark Westhusin (right) and Tae
Young (left) of the College of Veterinary
Medicine at Texas A&M University on
Febuary 8, 2002.
Human Cloning Cont.
 Therapeutic cloning is one type of cloning that does
not intend to create a human being, but only to use
the cells derived from the nuclear transfer to harvest
stem cells [destroys the embryo <14 days old].
 Embryonic stem cells were first isolated from mouse embryos in
the 1980s.
 Human embryonic stem cells were not isolated until 1998.
 News
What is a Stem Cell?
 Stem cells are undifferentiated
(pluripotent) cells [capable of
differentiation] that can develop
in to more specialized cells; two
types:

Embryonic: from the inner cell mass of
an embryo [blastocyst stage - 4 days
post fertilization]
 Somatic: more rare, have been found
in cord blood, bone marrow, brain,
eyes, muscle, among others; often fail
to survive outside of the body
 Animation
Karim Si-Tayeb changes media for
stem cells while working in the
Duncan lab, where they are
producing and maintaining induced
pluripotent stem cells (iPS) and
embryonic stem cells (ES), 2008.
Rick Wood/McClatchy-Tribune
Photos.
http://stemcells.nih.gov/info/basics/pages/basics6.aspx
Stem Cells Cont.
 Stem cells have proposed applications –
Study human development
2. Study new medications [rather than testing animals
or humans]
3. Cell therapies [EX using stem cells to repair ailing
organs, treating diseases including Alzheimer's,
strokes, spinal cord injuries]
 Video
http://newsblog.mayoclinic.org/2012/11/29/stem-cell101-mayo-clinic-expert-answers-commonly-askedquestions/
1.
“In the 1980s, studies on monkeys and rats
showed that when fetal brain tissue rich
in stem cells was implanted into the brains of
diseased animals, there was a regeneration of
functional brain cells and a reduction or
elimination the symptoms of the disease.”
"Stem Cells."
http://ic.galegroup.com/ic/scic/ImagesDetailsPage/ImagesDetailsWin
dow?total=15&query=BS+stem+cells&prodId=SCIC&windowstate=nor
mal&mode=view&limiter=AC+y&displayGroupName=Images&currPa
ge=1&sortBy=relevance%2Cdescending&action=e&catId=&view=doc
Display&documentId=GALE%7CPC4205136772
Embryonic stem cells (ESCs) are
shown using a colored scanning
electron micrograph (SEM). Research
using ESCs is controversial as it
requires the destruction of an
embryo. Magnification: x3000 when
printed at 10 centimeters wide.
© Steve Gschmeissner/SPL/Getty
Images.
Citations
"Clone and Cloning." The Gale Encyclopedia of Science. Ed. K. Lee Lerner and Brenda Wilmoth Lerner. 4th ed. Vol. 2. Detroit:
Gale, 2008. 959-962. Gale Virtual Reference Library. Web. 21 Jan. 2013.
"Cloning, Human." Biotechnology: Changing Life Through Science. Vol. 1: Medicine. Detroit: U*X*L, 2007. 65-69.Gale Virtual
Reference Library. Web. 22 Jan. 2013.
"Embryonic stem cells." Biotechnology: In Context. Ed. Brenda Wilmoth Lerner and K. Lee Lerner. Detroit: Gale, 2012. In
Context Series. Gale Science In Context. Web. 22 Jan. 2013.
"Introduction to Should the Government Fund Embryonic Stem Cell Research?: At Issue." Should the Government Fund
Embryonic Stem Cell Research? Ed. Amy Francis. Detroit: Greenhaven Press, 2009. At Issue. Gale Opposing Viewpoints
In Context. Web. 22 Jan. 2013.
"Public opinion about embryonic stem cell research, 2001-10." Genetics and Genetic Engineering. Barbara Wexler. 2011 ed.
Detroit: Gale, 2010. Information Plus Reference Series. Gale Opposing Viewpoints In Context. Web. 22 Jan. 2013.
"Stem Cell Research." MCT Photos. McClatchy-Tribune Information Services, 2010.Gale Opposing Viewpoints In Context. Web.
22 Jan. 2013.
"Stem Cells." The Gale Encyclopedia of Science. Ed. K. Lee Lerner and Brenda Wilmoth Lerner. 4th ed. Vol. 5. Detroit: Gale,
2008. 4162-4164. Gale Virtual Reference Library. Web. 22 Jan. 2013.
"Therapeutic Cloning." Biotechnology: Changing Life Through Science. Vol. 1: Medicine. Detroit: U*X*L, 2007. 267-270. Gale
Virtual Reference Library. Web. 22 Jan. 2013.
"Wanted: Neanderthal Clone Baby Geneticist Seeks Adventurous Surrogate Mother." International Business Times - US ed. 21
Jan. 2013.Infotrac Newsstand. Web. 22 Jan. 2013.
De Stasio, Elizabeth A. "Cloning Organisms." Genetics. Ed. Richard Robinson. Vol. 1. New York: Macmillan Reference USA,
2003. 161-165. Gale Virtual Reference Library. Web. 22 Jan. 2013.
Hoyle, Brian Douglas. "Stem Cell Cloning." Biotechnology: In Context. Ed. Brenda Wilmoth Lerner and K. Lee Lerner. Detroit:
Gale, 2012. In Context Series. Gale Science In Context. Web. 21 Jan. 2013.
Lewis, Ricki. "Cloning: Ethical Issues." Genetics. Ed. Richard Robinson. Vol. 1. New York: Macmillan Reference USA, 2003. 158161. Gale Virtual Reference Library. Web. 22 Jan. 2013.
Magner, Lois N., Stephanie Watson, and Elaine Wacholtz. "At this stage of our knowledge, are claims that therapeutic cloning
could be the cure for diseases such as diabetes and Parkinson's premature and misleading." Science in Dispute. Vol. 2.
Detroit: Gale, 2002. 227-236. Gale Virtual Reference Library. Web. 22 Jan. 2013.
McGee, Glenn. "Human Cloning." Encyclopedia of Science, Technology, and Ethics. Ed. Carl Mitcham. Vol. 2. Detroit:
Macmillan Reference USA, 2005. 938-942. Gale Virtual Reference Library. Web. 22 Jan. 2013.