Biotechnology
Chapter 20
What is Cloning?
 Cloning is the creation of an organism that is an exact genetic
copy of another. This means that every single bit of DNA is the
same between the two!
What animals have been cloned?
 In 1952, the first animal, a tadpole, was cloned.
 Before the creation of Dolly, the first mammal cloned from the cell of an
adult animal, clones were created from embryonic cells.
 Since Dolly, researchers have cloned:
 Sheep, goats, cows, mice, pigs, cats, rabbits, and a gaur.
 All these clones were created using nuclear transfer technology.
 Some species may be more resistant to somatic cell nuclear transfer than
others.
 Traumatic experience , and improvements in cloning technologies may be
needed before many species can be cloned successfully.
Types of Cloning
 Somatic Cell Nuclear Transfer
(Reproductive cloning)
 Recombinant DNA Technology or DNA Cloning
 Therapeutic cloning
Somatic Cell Nuclear Transfer (SCNT)
 Produces an exact clone, or genetic copy, of an individual.
 Method used to create Dolly the Sheep.
 Somatic cell: Any cell in the body other than the gametes. In
mammals, every somatic cell has two complete sets of
chromosomes (diploid)
Somatic Cell Nuclear Transfer (SCNT) &
Dolly (Reproductive Cloning )
 Researchers isolated a somatic cell from an adult female sheep.
 Next, they transferred the nucleus from that cell to an egg cell
from which the nucleus had been removed.
 The egg cell, with its new nucleus, was behaving just like a
fertilized zygote.
 It developed into an embryo, which was implanted into a surrogate
mother and carried to term.
Creating Dolly
Stage 1
Cell collected from a sheep’s udder.
Stage 2
Nucleus is removed from unfertilized egg of
second sheep.
Stage 3
Udder cell is inserted into egg with no
nucleus.
Stage 4
Insertion is successful.
Stage 5
Electrical charge is supplied.
Stage 6
Cells begin to divide.
Stage 7
Embryo implanted into a surrogate
Celebrity Sheep Died at Age 6
 Dolly was put down by lethal injection Feb. 14, 2003.
 Prior to her death, Dolly had been suffering from
lung cancer and crippling arthritis.
 Although most Finn Dorset sheep live to be 11 to 12
years of age, postmortem examination of Dolly
seemed to indicate that, other than her cancer and
arthritis, she appeared to be quite normal.
 The unnamed sheep from which Dolly was cloned had
died several years prior to her creation.
 Dolly was a mother to six lambs, bred the oldfashioned way.
Is cloning an organism the same as
cloning a gene?
 Cloning an animal, or any other organism, refers to
making an exact genetic copy of that organism.
 Cloning a gene means isolating an exact copy of a single
gene from the entire genome of an organism.
 Involves copying the DNA sequence of that gene into a
smaller, more accessible piece of DNA, such as a plasmid.
This makes it easier to study the function of the individual
gene in the laboratory.
Animation
Recombinant DNA Technology or DNA
Cloning
 Transfer of a DNA fragment from one organism to a self-
replicating genetic element such as a bacterial plasmid.
 The DNA is then be propagated in a foreign host cell.
 Been around since the 1970s, and it has become a common
practice in molecular biology labs today.
Plasmids
 Self-replicating extra-chromosomal circular DNA molecules
 Used by Human Genome Project (HGP) researchers to copy
genes
To "clone a gene"
 A DNA fragment containing the gene of interest is isolated from
chromosomal DNA using restriction enzymes and then united with a
plasmid that has been cut with the same restriction enzymes.
 When the fragment of chromosomal DNA is joined with its cloning
vector in the lab, it is called a "recombinant DNA molecule."
 Following introduction into suitable host cells, the recombinant DNA
can then be reproduced along with the host cell DNA.
Restriction Enzymes
 Aka: restriction endonucleases
 DNA-cutting enzymes
 Found in bacteria & made exclusively by prokaryotes.
 In order to sequence DNA, it is first necessary to cut it into smaller
fragments.
 A restriction enzyme recognizes and cuts DNA only at a particular sequence
of nucleotides.
 Originally discovered through their ability to break down, or restrict, foreign
DNA.
 Can distinguish between the DNA normally present in the cell and foreign
DNA
 Ie. infecting bacteria virus DNA (bacteriophage).
 Defend the cell from invasion by cutting the foreign DNA into pieces,
thereby rendering the DNA nonfunctional.
Restriction Enzymes
 Many restriction enzymes cut in an offset fashion.
 The ends of the cut have an overhanging piece of single-stranded
DNA. Known as "sticky ends"
 They are able to form base pairs with any DNA molecule that
contains the complementary sticky end.
 The union can be made permanent by DNA ligase, that forms
covalent bonds along the backbone of each strand.
 The result is a molecule of recombinant DNA (rDNA).
 Animation
Restriction Enzymes & Nucleases
 Nuclease -any enzyme that cuts the phosphodiester bonds of the DNA
backbone
 bond between a two sugar groups and a phosphate group
 Endonuclease -an enzyme that cuts some where within a DNA molecule.
 Exonuclease- cuts phosphodiester bonds by starting from the free end of
the a single DNA strand in the 3' to 5' direction
Restriction Enzymes
 Animations
Palindrome DNA
 A sequence of DNA which reads the same on both strands
 Ie. a palindrome).
 Such sequences often form the recognition sites for enzymes such as
restriction endonucleases.
Restriction Enzyme EcoR1
 EcoRI, one of many restriction enzymes, is obtained from the bacteria
Escherichia coli.
Benefits of Recombinant DNA
 Revolutionized genetics &
biotechnology industry
 Examples:
 Human insulin
 Human factor VIII (for males
with hemophilia A),
Other Cloning Vectors
 Bacterial plasmids
 Viruses
 Bacteria artificial chromosomes (BACs)
 Yeast artificial chromosomes (YACs)
 Cosmids are artificially constructed cloning vectors for infection
into E. coli cells.
 Bacteria are most often used as the host cells for recombinant DNA
molecules, but yeast and mammalian cells also are used.
Therapeutic Cloning
 Aka: "embryo cloning“
 Production of human embryos for use in research.
 Goal is not to create cloned human beings, but rather to
harvest stem cells that can be used to study human
development and to treat disease.
Animation
Can organs be cloned for use in
transplants?
 To do this, DNA would be extracted from the person in need of a
transplant and inserted into an enucleated egg.
 After the egg containing the patient's DNA starts to divide, embryonic
stem cells that can be transformed into any type of tissue would be
harvested.
 The stem cells would be used to generate an organ or tissue that is a
genetic match to the recipient.
 In theory, the cloned organ could then be transplanted into the patient
without the risk of tissue rejection.
Many challenges must be overcome before
"cloned organ" transplants become reality.
 More effective technologies for creating human embryos, harvesting stem
cells, and producing organs from stem cells would have to be developed.
 In 2001, scientists cloned the first human embryos; however, the only
embryo to survive the cloning process stopped developing after dividing into
six cells.
 In 2002, scientists successfully transplanted kidney-like organs into cows.
The team of researchers created a cloned cow embryo by removing the DNA
from an egg cell and then injecting the DNA from the skin cell of the donor
cow's ear. Since little is known about manipulating embryonic stem cells
from cows, the scientists let the cloned embryos develop into fetuses. The
scientists then harvested fetal tissue from the clones and transplanted it into
the donor cow. In the three months of observation following the transplant,
no sign of immune rejection was observed in the transplant recipient.
What are the risks of cloning?
 Reproductive cloning is expensive and highly inefficient.
 More than 90% of cloning attempts fail to produce viable offspring.
 More than 100 nuclear transfer procedures could be required to
produce one viable clone.
 Cloned animals tend to have more compromised immune function
 Clones have been known to die mysteriously.
 Example, Australia's first cloned sheep appeared healthy and energetic on
the day she died, and the results from her autopsy failed to determine a
cause of death.
More Risks
 Programming errors in the genetic material from a donor cell.
 When an embryo is created from the union of a sperm and an egg,
the embryo receives copies of most genes from both parents.
 A process called "imprinting" chemically marks the DNA from the
mother and father so that only one copy of a gene (either the
maternal or paternal gene) is turned on.
 Defects in the genetic imprint of DNA from a single donor cell may
lead to some of the developmental abnormalities of cloned embryos
PCR
 Purpose: Used to identify and amplify (copy)DNA in a sample
 Makes millions of copies of a gene
 Necessary to have enough starting template for sequencing.
Benefits
 Takes only approximately 2 hours
 Bypasses the need to use bacteria for amplifying DNA.
 Relatively simple and inexpensive
 Used every day to diagnose diseases, identify bacteria and viruses,
match criminals to crime scenes, and in many other ways
 Animation………….Animation
Steps of PCR
 Pair of primers (short single-stranded DNA segments) are needed that
attach to either side of the target DNA to be copied.
 The target DNA, the primers, a polymerase molecule (heat resistant)which
duplicates DNA, and a supply of nucleotides are mixed together are go
through a series of heating and cooling cycles.
 After approximately 30 cycles, the segment will have been copied 250
million times.
 The results of PCR can then be analyzed in various ways, such as agarose gel
electrophoresis or sequencing.
Enzymes & Primers
 Primers:
 About 20 nucleotides long
 Complementary to the beginning and end of the DNA fragment of
interest which one needs to amplify.
 Select the boundaries of the region to be amplified by PCR.
 During the PCR annealing cycle, PCR primers anneal to the
complementary region of the DNA.
 Anneal-to heat and then cool in order to separate strands and induce
combination at lower temperatures especially with complementary strands
Heating & Cooling Process
 1st Heating Process
 Samples heated to 95 degrees C
 Purpose: Denature DNA- breaking bonds
 2nd Cooling Process (Annealing)
 60 degrees C
 Allows primers to form Hydrogen bonds or anneals with complementary
DNA strand
Heating & Cooling Process
 3rd Heating Process
 Temperature raised to 72 degrees C
 Taq polymerase(optimal temperature) begins polymerization adding
nucleotides to the 3’end of each primer attached to a DNA strand
 Animation
The segment of the target DNA between the primers will
be copied during each cycle, accumulating in the mixture
in an exponential fashion.
 After 1st complete cycle-----2 double stranded copies of the target
DNA
 After 2nd complete cycle----- 4 copies
 After 3rd complete cycle----- 8 copies
 After 25 cycles- 33 million copies
Steps of PCR
PCR
Steps of PCR
Enzymes & Primers
 Taq polymerase
 Obtained from hot spring bacteria
 Able to withstand extreme PCR heat
Gel electrophoresis.
 When restrictive endonuclease is used the DNA split to multiple
mixed fragments, they are then separated by gel electrophoresis.
 The gel is then blotted on nitrocellulose paper.
 Turns to a solid form
 Now we have separate solid DNA fragments.
 A probe is then added to bind to its matching fragment which can
then be visualized by autoradiography.
 Animation
Types of Blotting
 If the substance is DNA it is termed Southern Blotting.
 If the substance is RNA it is termed Northern blotting.
 If the substance is protein it is termed Western blotting.
Purpose of PCR & Gel Electrophoresis
 PCR
 Amplifying a section of DNA
 Gel Electrophoresis
 Sort DNA by size
 Read the sequence of a DNA fragment
DNA Arrays
 Microscopic spot containing identical single-stranded molecules
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

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of DNAs, usually oligonucleotides or complementary DNAs
Analyze patterns of gene expression.
DNA microarrays often consist of glass slides with spots of
attached DNA fragments.
The DNA fragments act as probes for specific sequences in a
sample.
Animation …………. Animation
Stem Cells
 Important to biomedical researchers because they can be used to
generate virtually any type of specialized cell in the human body.
 Extracted from the egg after it has divided for 5 days.
 The egg at this stage of development is called a blastocyst.
 The extraction process destroys the embryo
 Raises ethical concerns.
 Hope that one day stem cells can be used to serve as replacement
cells to treat heart disease, Alzheimer's, cancer, and other diseases.
 Animation
Pluripotent
 Refers to a stem cell that has the potential to differentiate into
any of the three germ layers:
 Endoderm (interior stomach lining, gastrointestinal tract, the
lungs)
 mesoderm (muscle, bone, blood)
 Ectoderm (epidermal tissues and nervous system).
 Pluripotent stem cells can give rise to any fetal or adult cell type.
 However, alone they cannot develop into a fetal or adult animal
because they lack the potential to contribute to extraembryonic
tissue, such as the placenta.
Totipotent
 One of the most important stem cells types because they have the
potential to develop into any cell found in the human body.
 In human development, the egg cell in a woman and the sperm cell
from a man fuse together to form a single cell called the zygote.
The zygote divides numerous times and forms cells that are the
precursors to the trillions of cells that will eventually constitute the
human body.
Practical applications of DNA
Technology
 Gene therapy
 DNA transformation Animation Animation
 GMO
Genomic library
 DNA library is a collection of cloned DNA fragments. There are two
types of DNA library:
 The genomic library contains DNA fragments representing the
entire genome of an organism.
 The cDNA library contains only complementary DNA molecules
synthesized from mRNA molecules in a cell.
 The advantage of cDNA library is that it contains only the coding region of
a genome
 cDNA libraries can be used to clone cDNA for a known gene to discover
the sequence of the mRNA it encodes
Use of complementary DNA (cDNA)
 Doublestranded DNA version of an mRNA molecule.
 In eukaryotes, an mRNA is a more useful predictor of a polypeptide
sequence than is a genomic sequence, because the introns have been
spliced out.
 Researchers prefer to use cDNA rather than mRNA itself because
RNAs are inherently less stable than DNA and techniques for
routinely amplifying and purifying individual RNA molecules do not
exist.
 Animation
cDNA