Recombinant DNA and Genetic Engineering

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Recombinant DNA and Genetic Engineering

• Chapter 16

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Impacts, IssuesGolden Rice or Frankenfood?

• Scientists created transgenic rice (Golden Rice)
  as a vitamin A supplement for undernourished
  nations is the benefit worth the risk in these
  gene-manipulated food sources?

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16.1 Cloning DNA

• Process to add genes to food or other cell types
  is simple in principle
• Researchers cut up DNA from different sources,
  then paste the resulting fragments together
• Cloning vectors can carry foreign DNA into host
  cells

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Cut and Paste for New DNA Combos

• Restriction enzymes
• Bacterial enzymes that cut DNA wherever a
  specific nucleotide sequence occurs
• Single-stranded DNA tails produced by the same
  restriction enzyme base-pair together
• DNA ligase bonds sticky ends together
• Recombinant DNA
• Composed of DNA from two or more organisms

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Making Recombinant DNA
This is especially useful to introduce genes into
a sequence in research.
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DNA Cloning

• DNA cut into fragments by restriction enzymes is
  inserted into cloning vectors (plasmids) cut with
  the same enzyme
• Cloning vectors with foreign DNA are placed in
  host cells, which divide and produce many clones,
  each with a copy of the foreign DNA

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Cloning Vectors
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DNA Cloning
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cDNA Cloning

• Complementary DNA (cDNA)
• DNA made from an mRNA template
• Reverse transcriptase transcribes mRNA to DNA,
  forming a hybrid molecule
• DNA polymerase builds a double-stranded DNA
  molecule that can be cloned
• Especially useful to obtain DNA without introns.

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cDNA Cloning by Reverse Transcriptase
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16.1 Key Concepts DNA Cloning by Lab and Plasmids

• Researchers routinely make recombinant DNA by
  cutting and pasting together DNA from different
  species
• Plasmids and other vectors can carry foreign DNA
  into host cells

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Genomes and DNA Libraries

• Genome
• The entire set of genetic material of an organism
• DNA libraries are sets of cells containing
  various cloned DNA fragments
• Genomic libraries (all DNA in a genome)
• cDNA libraries (all active genes in a cell)

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Probes Used for ID of DNA

• Probe
• A fragment of DNA labeled with a tracer
• Used to find a specific clone carrying DNA of
  interest in a library of many clones
• Nucleic acid hybridization
• Base pairing between DNA from different sources
• A probe hybridizes with the targeted gene

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Big-Time DNA Amplification PCR

• Polymerase chain reaction (PCR)
• A cycled reaction that uses a heat-tolerant form
  of DNA polymerase (Taq polymerase) to produce
  billions of copies of a DNA fragment
• This is how a single drop of blood at a crime
  scene can become expanded to enough to make
  necessary tests and still be available for future
  testing if needed

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PCR in Overview

• DNA to be copied is mixed with DNA polymerase,
  nucleotides and primers that base-pair with
  certain DNA sequences
• Cycles of high and low temperatures break and
  reform hydrogen bonds between DNA strands,
  doubling the amount of DNA in each cycle

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PCR Steps to More
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16.2 Key Concepts Needles in Haystacks

• Researchers manipulate targeted genes by
  isolating and making many copies of particular
  DNA fragments

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16.3 DNA Sequencing

• DNA sequencing reveals the order of nucleotide
  bases in a fragment of DNA

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DNA Sequencing

• DNA is synthesized with normal nucleotides and
  dideoxynucleotides tagged with different colors
• When a tagged base is added, DNA synthesis stops
  fragments of all lengths are made
• Electrophoresis separates the fragments of DNA,
  each ending with a tagged base, by length
• Order of colored bases is the sequence of DNA
• Finished sequence is basis for comparison

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DNA Sequencing
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16.4 DNA Fingerprinting

• One individual can be distinguished from all
  others on the basis of DNA fingerprints
• Confidence here in results is extremely high, in
  the usually stated range of one in many millions

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DNA Fingerprints

• DNA fingerprint
• A unique array of DNA sequences used to identify
  individuals
• Short tandem repeats (STRs)
• Many copies of the same 2- to 10-base-pair
  sequences in a series along a chromosome
• Types and numbers of STRs vary greatly among
  individuals

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Creating DNA Fingerprints

• PCR is used to amplify DNA from regions of
  several chromosomes that have STRs
• Electrophoresis is used to separate the fragments
  and create a unique DNA fingerprint
• DNA fingerprints have many applications
• Legal cases, forensics, population studies

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DNA Fingerprints Forensics Case Example
You are on the jury. You are shown this prepared
comparison of DNA fingerprints, with ID as
shown. See if you can match suspect with sample
from the crime scene.
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16.3-16.4 Key Concepts Deciphering DNA Fragments

• Sequencing reveals the linear order of
  nucleotides in a fragment of DNA
• A DNA fingerprint is an individuals unique array
  of DNA sequences

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16.5 Studying Genomes

• Comparing the sequence of our genome with that of
  other species is giving us insights into how the
  human body works
• You already know of 98 percent same human
  sequences with that of chimpanzes
• How about 49 percent the same between a banana
  and a human?

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The Human Genome Project

• Automated DNA sequencing and PCR allowed human
  genome projects to sequence the 3 billion bases
  in the human genome
• 28,976 genes have been identified, but not all of
  their products or functions are known
• As of 2010, distinct gene numbers down to about
  23,000 by best estimates from work

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Sequencing the Human Genome
Computers have greatly speeded process up and
also increased accuracy.
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Genomics is a Growing Application

• Genomics The study of genomes
• Structural genomics
• Comparative genomics
• Analysis of the human genome yields new
  information about genes and how they work
• Applications in medicine and other fields
• Example APOA5 mutations and triglycerides

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DNA Chips Have a Future

• DNA chips
• Microarrays of many different DNA samples
  arranged on a glass plate
• Used to compare patterns of gene expression among
  cells of different types or under different
  conditions
• May be used to screen for genetic abnormalities,
  pathogens, or cancer

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16.6 Genetic Engineering

• Genetic engineering
• A laboratory process by which deliberate changes
  are introduced into an individuals genome
• Todays most common genetically modified
  organisms are bacteria and yeast
• Are used in research, medicine, and industry
• Example production of human insulin

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GMOs Now and Later

• Genetically modified organisms (GMOs)
• Individuals containing modified genes from the
  same species or a different species
• Future will have major control problems as the
  developer of GMO usually patents process/result
• Transgenic organisms
• Individuals containing genes transferred from a
  different species (also GMOs)
• Example Bacteria with jellyfish genes

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16.7 Designer Plants by GM

• Genetically engineered crop plants are widespread
  in the United States
• But can their designed change(s) jump to other
  plant life or end up incorporated in animals
  eating the modified plants?

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The Ti Plasmid a GMO Mechanism

• Ti plasmid
• Plasmid of bacteria Agrobacterium tumefaciens
• Contains tumor-inducing (Ti) genes
• Used as a vector to transfer foreign or modified
  genes into plants, including some food crops

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Ti Plasmid Transfer Steps
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Genetically Engineered Plants

• Crop plants are genetically modified to produce
  more food at lower cost
• Resistance to disease or herbicides
• Increased yield
• Plants that make pesticides (Bt protein gene)
• Drought resistance

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GMO Controversies

• 73 GMO crops are approved for use in US, with
  hundreds more pending
• Corn, sorghum, cotton, soy, canola, alfalfa
• Big problem of just a few companies doing nearly
  all the research and manufacturing can lead to
  a monopoly problem in future
• Facts and controversy real life
• In crops engineered for herbicide resistance,
  weeds are becoming resistant to herbicides
• Engineered genes are spreading into wild plants
  and nonengineered crops

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Some Genetically Modified Plants
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16.8 Biotech Barnyards

• Animals that would be impossible to produce by
  traditional breeding methods are being created by
  genetic engineering
• This can be really good for endangered animals
• Genetically engineered animals are used in
  research, medicine, and industry

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Of Mice and Men

• 1982 The first transgenic animals mice with
  genes for rat growth hormone

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Examples of Transgenic Animals

• Genetically modified animals are used as models
  of many human diseases
• Mice used in knockout experiments
• Genetically modified animals make proteins with
  medical and industrial applications
• Goats and rabbits that make human proteins
• Farms animals with desirable characteristics

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Some Genetically Modified Animals
That silly-looking featherless chicken is easily
the most commercially viable possibility shown
here. It would eliminate a costly part of chicken
processing and could enable very warm climate
poultry farms.
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Knockout Cells and Organ Factories

• Transgenic pigs with human proteins are a
  potential source of organs and tissues for
  transplants in humans
• May prevent rejection by immune system
• Xenotransplantation
• Transplantation of a tissue or organ from one
  species to another
• Pig heart valves used for many years.

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16.10 Modified Humans?

• We as a society continue to work our way through
  the ethical implications of applying new DNA
  technologies
• The manipulation of individual genomes continues
  even as we are weighing the risks and benefits of
  this research

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Gene Therapy Helping the Individual

• Gene therapy
• Transfer of recombinant DNA into body cells to
  correct a genetic defect or treat a disease
• Viral vectors or lipid clusters insert an
  unmutated gene into an individuals chromosomes
• Examples Cystic fibrosis, SCID-X1

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Getting Better by Gene Therapy

• 1998 A viral vector was used to insert unmutated
  IL2RG genes into boys with severe combined
  immunodeficiency disease (SCID-X1) most
  recovered immune function

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Getting Worse by Gene Therapy

• No one can predict where a virus-injected gene
  will insert into a chromosome several boys from
  the SCID-X1 study developed cancer
• In other studies, severe allergic reactions to
  the viral vector itself have resulted in death

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Getting Perfect Over Time

• Eugenic engineering
• Engineering humans for particular desirable
  traits, not associated with treatment of
  disorders

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16.6-16.10 Key Concepts Using the New
Technologies of GM

• Genetic engineering, the directed modification of
  an organisms genes, is now used in research, and
  it is being tested in medical applications
• Many questions must be answered about the ethics
  and consequences of manipulating the human genome
  some of these can be answered by our government
  but many will remain answerable only by the
  individual as he or she agrees to their usage
  personally.