Recombinant DNA Technologies

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Recombinant DNA Technologies The Age of
Genomics
From Chapters 19 22 Human Genetics Concepts
and Applications 6th edition By Ricki Lewis
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Recombinant DNA Technology

• A technique used to cut apart and splice together
  pieces of DNA from different sources
• Foreign DNA transferred into another cell may be
  produced along with substances coded for by the
  native genetic material of the cell or organism.
• Cells may become "factories" for the production
  of the protein coded for by the inserted DNA.
• It is possible to create genetically modified
  organisms because of the universal nature of the
  genetic code.
• Example Human gene for insulin was placed in
  bacteria to produce insulin in large quantities
  for diabetics. These bacteria are recombinant
  organisms.

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Recombinant DNA Technology Method Overview

• DNA is purified from cells or tissues
• Enzymes are used to cut DNA molecules and join
  them together
• DNA is transferred to a host cell
  (transformation)
• DNA replicated in cells
• If appropriate sequences are present, gene may be
  transcribed and translated to protein

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Creating recombinant DNA molecules

• The DNA of interest (target) is cut into pieces
  with a restriction enzyme.
• The vector DNA is also cut with the same
  restriction enzyme.
• Vector DNA is a shuttle for moving and copying
  the DNA fragment of interest.
• The cut target is combined with the cut vector
• The complementary ends of the DNAs bind and DNA
  ligase reattaches the sugar-phosphate backbone of
  the DNA.

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Vectors

• DNA molecules that can be moved into and
  replicated in an organism. They are shuttles for
  moving and copying another DNA fragment or gene.
  
• classified by the organisms which replicated the
  vector and the size of DNA that can be inserted

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Creating recombinant DNA molecules
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Recombinant DNA
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Recombinant DNAs are propagated as clones in each
colony of bacteria.
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Creating recombinant DNA molecules

• Recombinant DNA molecule (vector and insert) is
  introduced into the host organism. Introduction
  methods focus on getting DNA across the membrane
  of the host cell
• Electroporation electricity opens
  a temporary hole
• Microinjections microscopic
  needles inject DNA
• Liposomes fatty bubbles
  move across membranes
• Particle bombardment DNA coated bullets shot
  into cell
• Chemicals salts open a
  temporary hole
• Viruses DNA or RNA
  viruses infect cell

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Injecting DNA into a plant cell
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Selecting recombinant molecules

• Three types of cells can result from attempt to
  introduce a DNA molecule into a bacterial cell
• Cells without vector
• Cells with vector with no inserted DNA
• Cells with vector with inserted DNA

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Selecting for cells with vectors

• Vectors are commonly engineered to carry
  antibiotic resistance genes.
• Host bacteria die in the presence of the
  antibiotic.
• Bacteria harboring the vector survive.
• Growing cells on media with antibiotics ensures
  that all growing cells must carry the vector.

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Selecting cells with inserted DNA

• The site of insertion of the DNA of interest can
  be within a gene on the vector. Insertion of a
  DNA fragment will disrupt the vector gene.
• The vector gene LacZ produces an enzyme which
  allows the bacteria to turn blue in the presence
  of certain media.
• Insertions in the lacZ gene prevent lacZ enzyme
  production and the bacteria are white.
• Bacteria with vector that are white carry an DNA
  inserted in the lacZ gene.
• RARE mutations in the lacZ gene will also make
  white bacteria.

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Genetic map of the phasmid pUC
unique cloning
sites
virus ori
E. coli ori
Antibiotic resistance
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• Genomic DNA libraries.
• Contains clones representative of the entire
  nuclear genome, including expressed sequences,
  regulatory/promoters, constitutive and
  non-functional DNA.
• Within an organism all nucleated cells contain
  essentially the same DNA genomic libraries made
  from different cells or tissues are
  indistinguishable.
• Genomic libraries can be constructed in any
  vector but are most efficiently made in phage,
  cosmid or artificial chromosome vectors.

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What is a cDNA?

• Complementary DNA (to mRNAs)
• Copy of mRNA into DNA using enzyme reverse
  transcriptase
• Useful because all introns are spliced, leaving
  only coding region
• Using cloning method, cDNA libraries can be
  constructed

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• Complementary (cDNA) libraries.
• constructed from mRNA using RNA-dependant DNA
  polymerase (reverse transcriptase).
• contain only sequences present in mature mRNA.
• are different depending on source of cell
  type/tissue and time of development.
• usually constructed in plasmid vectors.
• cDNA libraries are the source libraries for
  expressed sequence tags (ESTs) in the GENBANK
  database.

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Construction of complementary DNA (cDNAs)
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Applications of recombinant DNA

• Recombinant DNA is used to
• Study the biochemical properties or genetic
  pathways of a protein
• Mass produce a particular protein
• Insulin is made in bacteria for diabetics.
• Renin, a calf stomach enzyme, is made in bacteria
  for cheese production.
• Human growth hormone is produced to treat
  pituitary dwarfism in children.
• Modify an organism
• Bt gene from Bacillus thuringiensis is a
  pesticide introduced into corn and soybeans.

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Expression cloning of normal gene products to
produce medically valuable gene products.

• Examples include
• Gene product Used to treat
• Blood clotting factors Hemophilia
• Erythropoietin Anemia
• Insulin Diabetes
• Growth Hormone GH deficiency
• various interferons infections
• Interleukin-2 Renal cell carcinoma
• DNase Cystic fibrosis

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Transgenics
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Transgenic organisms

• A transgenic organism is an organism that
  develops from genetically altered gametes.
• All of the cells of the organism will have the
  genetic alteration present.
• Future progeny of the transgenic organism will
  also carry the genetic alteration.

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Transgenics

• Genetically modified pigs were engineered to
  produce less polluting manure.

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Transgenic Pharming

• Pharming refers to the harvesting of proteins
  from secreted materials like milk.
• Transgenic sheep and goats have been created to
  produce human proteins for the drug industry.

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Gene targeting

• Is a method in which a specific gene is altered.
  
• A knocked out gene is one that is removed by
  deletion or mutation.
• A knocked in gene is one in which a specific
  segment is replaced by a desired mutation.

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Gene targeting

• Homologous recombination is a natural process in
  which DNA sequences which are similar or
  identical recombine (mechanism of crossing over).
• Introduction of homologous DNA flanking an
  insertion in a vector allows homologous
  recombination to occur.

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Gene targeting
Disruption of a pigment gene occurs by homologous
recombination in an embryonic stem cell (ES
cell). ES cells carrying the insertion are
injected into blastocysts and placed in female
mice. Mosaic progeny have cells derived from the
host blastocysts and the injected ES
cells. Gametes carrying the insertion will
create progeny in which all cells carry the
alteration.
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Knockout mutations in mice may create a model
system for studying human disease

• Knockout of the neurofibromatosis type I gene
  (NF1) in the mouse on the left leads to tumor
  formation.
• Studying NF1 in humans is difficult due to
  lethality in early development.

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Normal knockouts

• Surprisingly, some genes can be knocked out and
  have no apparent effect on phenotype.
• Explanations
• Other genes encode the same or similar proteins
  which can compensate (redundancy)
• Absent protein does not cause an effect but an
  abnormal protein might
• Gene may have no function or different role than
  hypothesized
• Conditions required to observe phenotype are not
  present.
• Need of X collagen in skeletal repair is only
  observable in conditions in which fractures occur.

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Recombinant technologies and ethics

• 1973 1978. Recombinant DNA technology and
  safety were major concerns with legislation to
  prohibit certain technologies
• 1982 present. Almost no recombinant DNA
  technologies are prohibited.

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GMOs

• Genetically Modified Organisms
• GM are technologies that alter the genetic makeup
  animals, plants, or bacteria
• An organism created using recombinant DNA
  technology may be called
• genetically modified,
• genetically engineered, and/or
• transgenic

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GMOs

• 167 million acres grown by 7 million farmers in
  18 countries were planted with transgenic crops
  (2003)
• herbicide- and insecticide-resistant soybeans,
  corn, cotton, and canola
• sweet potato resistant to a virus threatening the
  African harvest
• rice with increased iron/vitamins to alleviate
  chronic malnutrition in Asia
• plants able to survive weather extremes
• Countries that grew 99 of the global transgenic
  crops were (in 2003)
• United States (63)
• Argentina (21)
• Canada (6)
• Brazil (4)
• China (4)
• South Africa (1)

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On the GMO horizon

• bananas that produce human vaccines against
  infectious diseases - hepatitis B
• fish that mature more quickly
• fruit and nut trees that yield years earlier
• plants that produce new plastics with unique
  properties.

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GMO risks

• Safety Potential human health impact
• Allergens
• transfer of antibiotic resistance markers
• Other unknown effects
• Safety Potential environmental impact
• unintended transfer of transgenes through
  cross-pollination
• unknown effects on other organisms (e.g., soil
  microbes)
• loss of flora and fauna biodiversity
• Access and Intellectual Property
• Domination of world food production by a few
  companies
• Increasing dependence on Industralized nations by
  developing countries
• Biopiracyforeign exploitation of natural
  resources
• Ethics
• Violation of natural organisms' intrinsic values
• Tampering with nature by mixing genes among
  species
• Objections to consuming animal genes in plants
  and vice versa
• Stress for animals
• Labeling
• Not mandatory in some countries

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U R what U eat

• In US, it is not required that GM products are
  labeled
• All crop produces are genetically modified
• Can test and label foods as not produced through
  bioengineering
• Almost no GM foods in Europe, where strict
  labeling laws are also in effect

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GMO unlimited possibilities!
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Cloning eukaryotes
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Cloning of organisms

• Clones are cells or organisms arising from a
  single ancestor. Cloned cells or individuals are
  (almost) identical.
• Cloning of plants is usually based on somatic
  cell manipulation.
• Cloning of animals is based on manipulation of
  the nucleus in oocytes or early embryos.

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Plant Cloning
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Cloning from differentiated somatic cells.
Ian Wilmut and his team of researchers at Roslin
Institute in Scotland cloned Dolly, the first
mammal to be cloned from a somatic cell.
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Suction pipette
micropipette
A pronucleus being injected with DNA to produce a
transgenic mouse. This technique can also be used
to remove nuclei for transfer to enucleated
oocytes for cloning.
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Nuclear transfer method of producing clones
Animal Cloning
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Four reasons why genetically identical animals
are not phenotypically identical.
Mitochondria Immune response Epigenetics Embryo
development
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Cloning at Texas AM

• World leader in producing the most number of
  clones from different species
• Cattle
• Second Chance (1999)
• 862 (2000)
• Goat Second Addition (2001)
• Pig litter (2001)
• Cat - Cc (2001)
• Deer Dewey (2003)
• Horse Paris Texas (2005)

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Ethical Issues

• Is human cloning ethical? Playing with nature?
• Does a clone have parents?
• Autonomy?
• Might cloned children be able to choose their own
  destiny?
• What about cloning children who have died at a
  young age?
• Would it matter if the death were accidental?
• Does cloning to create stem cells (therapeutic
  cloning) justify destroying a human embryo?

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The Human Genome Project

• The decade long project to sequence the human
  genome or determine the order of the nucleotides
  present in each of the chromosomes end to end.
• The draft of the human genome was announced in
  February 2001.
• This represents the work of thousands of
  researchers in an international collaboration.

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Human Genome Project uses of genetic information
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The Human Genome Project

• Before sequencing the human genome, a framework
  map of the genome was needed.
• Correspondences between cytogenetic information,
  genetic linkage information, and physical
  information was determined.

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Two methods for sequencing the human genome
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Steps in genome sequencing and analysis

• Obtain chromosome maps with landmarks from
  linkage or cytogenetic studies or RFLP sites
• Obtain chromosome pieces in gene libraries or
  shotgun entire sequence
• Sequence the pieces
• Overlap aligned sequences
• Compare sequence to other species

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Overlapping sequence
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Overlapping sequence
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The International Human Genome Sequencing
Consortium published the first draft of the
human genome in the journal Nature in February
2001 with the sequence of the entire genome's
three billion base pairs some 90 percent
complete. Upon publication of the majority of
the genome, the genome could be thought of in
terms of a book with multiple uses "It's a
history book - a narrative of the journey of our
species through time. It's a shop manual, with
an incredibly detailed blueprint for building
every human cell. And it's a transformative
textbook of medicine, with insights that will
give health care providers immense new powers to
treat, prevent and cure disease."
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The existing and ultimate products of the HGP
will give the world a resource of detailed
information about the - structure, -
organization, and - function of the complete
set of human genes. This information can be
thought of as the basic set of inheritable
"instructions" for the development and function
of a human being.
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Insights Learned from the Sequence
The human genome contains 3164.7 million chemical
nucleotide bases (A, C, T, and G). The average
gene consists of 3000 bases, but sizes vary
greatly, with the largest known human gene being
dystrophin at 2.4 million bases. The total
number of genes is estimated at 30,000 to 35,000,
much lower than previous estimates of 80,000 to
140,000 that had been based on extrapolations
from gene-rich areas as opposed to a composite of
gene-rich and gene-poor areas. The order of
almost all (99.9) nucleotide bases are exactly
the same in all people. The functions are
unknown for over 50 of discovered genes.
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Less than 2 of the genome encodes for the
production of proteins. Repeated sequences that
do not code for proteins ("junk DNA") make up at
least 50 of the human genome. Repetitive
sequences are thought to have no direct
functions, but they shed light on chromosome
structure and dynamics. Over time, these repeats
reshape the genome by rearranging it, thereby
creating entirely new genes or modifying
and reshuffling existing genes. During the past
50 million years, a dramatic decrease seems to
have occurred in the rate of accumulation of
repeats in the human genome.
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The major areas that genetics will influence
include

• Identification of all human genes and their
  diversity (as well as in other animals and
  plants)
• Targeted gene therapy in humans (somatic cells)
• Clinical tests for hundreds of genetic attributes
  such as personality, intelligence, athletic
  ability, life span expectations, diseases, tissue
  and organ transplantation and others.

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Nonhuman genome projects

• What is the minimum set of genes required for
  life?
• Mycoplasma genitalium has 480 protein encoding
  genes, of which 265-350 are essential.

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Comparative Genomics

• What are the fundamental distinctions between the
  three domains of life?
• Archaea share fewer than half of their genes with
  other known organisms.
• The bacteria, E. coli, has 4,288 genes. One-third
  have unknown functions.

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Comparative Genomics

• What is the simplest organism with a nucleus?
• Even the unicellular yeast, Sacchromyces
  cervisiae had 5,885 genes including one-third
  which have human counterparts.

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Comparative Genomics

• What is the basic blueprint of an animal?
• Caenorhabditis elegans is a 959-celled nematode
  worm.
• Movement of each of its cells during development
  has been mapped. The 97 million base genome is
  sequenced and contains 18,425 genes.
• Many of the signaling pathways, cytoskeletal
  elements and brain proteins are very similar to
  human counterparts.
• Drosophila melanogaster is a fruit fly with
  13,601 genes.
• Of 289 human genes known to cause disease, 177
  have counterparts in the fruit fly.
• These organisms may make good model systems for
  understanding human genetics and disease.

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As we enter the post human genome project
period.

• Within the next 25 years technical capabilities
  of animal and plant molecular genetics will be
  the largest biological revolution in the history
  of mankind

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Is this revolution a dream come true or a
nightmare?

• Consider gene therapy for recessive lethal
  conditions in humans.
• Should the germ line cells also be corrected?
• If yes, who is in charge of how the human genome
  is manipulated?
• If no, what will happen to the frequency of these
  recessive alleles in subsequent generations?

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Theodosius Dobzhansky If we enable the weak
and deformed to live and promulgate their kind,
we face the prospect of a genetic twilight. But
if we let them die or suffer when we can help
them, we face the certainty of a moral twilight.