RECOMBINANT DNA TECHNOLOGY

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RECOMBINANT DNA TECHNOLOGY

• Syllabus section 11.4 / 12.6
• Textbook Reference Chapter 10 / 11

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Terms

• DNA
• Sequence of bases that codes for the production
  of proteins

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• Chromosome
• DNA packed in protein

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• Gene
• Part of the DNA sequence that codes for a
  single polypeptide / protein

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• Genome
• The whole package

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Human Genome Project

• Project goals are to
• identify all the approximate 30,000 genes in
  human DNA,
• determine the sequences of the 3 billion base
  pairs that make up human DNA,
• store this information in databases.

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Human chromosome number 1

• Total number of genes on chromosome 2430

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Recombinant DNA Technology

• Involves the combination of DNA from one organism
  with DNA from another organism.
• Inserted DNA is decoded in its new cell,
  producing the protein for which it codes.

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DNA

• UNIVERSAL CODE
• As the genetic code is the same in all organisms
  - a cell is unable to distinguish between genetic
  material from different sources.

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Why do it ?

• Medical applications
• Agricultural applications

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An example medical application

• Genetically engineered micro-organisms
• - human genes for insulin and growth
  hormone have been inserted into bacterial
  cells.
• The bacteria produces human hormones

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Both fungi and bacteria are used to produce
Insulin

• Yeast cells

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Examples agricultural application

• Genetically modified plants
• To improve resistance to
• pests
• diseases
• herbicides
• cold or drought

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• Produce salt-tolerant crops
• Produce plants that make pharmaceutical
  substances eg edible vaccines
• Improve the nutritional value higher vitamin
  levels

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• Biotech researchers at the Boyce Thompson
  Institute for Plant Research in Ithica, New York,
  are studying ways to fortify raw foods --
  including bananas and potatoes -- with vaccines
  to provide painless, inexpensive protection
  against disease.

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Insect infested cotton plants
Transgenic plant Normal plant
with a bacterial gene that

produces an insecticide
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Luciferase gene from a firefly was inserted into
a tobacco plant. When the plant was fed
luciferin the plant glowed in the dark !!!
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Further examples

• Genetically modified animals
• e.g. The human proteins, haemoglobin and blood
  clotting factors are produced in milk of
  transgenic cows, goats and sheep.
• Gene therapy

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Examples Gene therapy
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Recombinant DNA technology

• The procedure is divided into a number of stages

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Method

• 1. Isolate the gene.
• 2. Cut open a vector that will transfer the gene
  into a new cell.
• 3. Stick the gene into the cut vector.

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Method

• 4. Put the vector (containing the gene) into the
  new cell.
• 5. Make the cells multiply.
• 6. Isolate and purify the protein made by these
  cells.

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1. Isolate the Gene

• 3 different methods
• A - Working back from the protein

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• Find the amino acid sequence of the protein
• Use the genetic code to work out the base
  sequence
• Make DNA with that base sequence
• complementary DNA (cDNA)

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• B - Using messenger RNA
• Extract mRNA from a cell
• Use enzyme - reverse transcriptase - to make
  complementary DNA

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c DNA

• cDNA is a single strand
• Using DNA polymerase (the same enzyme that causes
  replication)
• A double strand of DNA is made

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• C - Using DNA probes
• Use a DNA probe to find the gene

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DNA Probe

• is used to find a single gene within a genome
• is a short, single strand of DNA that matches
  part of the base sequence in the gene
• is labelled with a radioactive or fluorescent
  marker

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• Cut the gene from the chromosome
• DNA is cut using a enzyme called
• a restriction endonuclease
• Simply known as a restriction enzyme

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Restriction Enzymes

• Cut DNA
• at specific base sequences
• many different types - each cutting at a
  different base sequence

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Restriction enzymes

• Extracted from bacteria
• Protect bacteria from viral attack by cutting up
  the invading viral DNA
• Named after the bacteria

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Restriction Enzyme

• Eg BamHI
• G G A T C C
• C C T A G G
• G G A T C C
• C C T A G G STICKY ENDS

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Restriction Enzyme

• Eg HindIII
• A A G C T T
• T T C G A A
• A A G C T T
• T T C G A A
  STICKY ENDS

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Restriction Enzyme

• Eg HpaI
• G T T A A C
• C A A T T G
• G T T A A C
• C A A T T G
• (no sticky ends)

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Sticky ends v non sticky ends!!!

• Some restriction enzymes produce staggered cuts
• This produces an unpaired strand of nucleotides
• A STICKY END

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Advantage of a sticky end?

• If another strand of DNA is cut by the same
  restriction enzyme.
• The two cut ends will be complementary.
• So they stick together easily

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Lets Review the Method!

• Recombinant DNA technology involves
• 1. Isolating the gene - v
• Next
• 2. Cut open a vector to transfer the gene into
  the new cell.

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Vectors remember.

• Vectors are carriers
• they carry the gene into cell.
• Certain viruses can be used.

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Virus vectors

• Viruses normally inject their genetic material
  into cells
• They will do this with any DNA inserted into the
  virus

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Virus vector
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• One common type of vector is a Plasmid

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Plasmids

• Plasmids are small, circular, double strands of
  DNA
• Drawn like this

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• Plasmids are found in bacteria.
• They carry non essential codes
• Eg. Antibody resistance
• They can pass from one cell to another

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Bacterial structure
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A Plasmid
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2. Cut open the vector

• The plasmid is cut open using the same
  restriction enzyme that was used to cut the gene
  from the DNA strand.
• This gives the same sticky ends.

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• Mix the cut gene and cut plasmid together
• Sticky ends of the gene and sticky ends of the
  plasmid join.

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• Complementary base pairs come together
• The plasmid and the gene fit together
• BUT they are only held by weak hydrogen bonds
  between the bases

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3. Stick gene into vector

• A condensation reaction is needed to attach the
  phosphate / sugar backbones
• This requires an enzyme
• DNA ligase

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Inserting a gene into a vector
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• The new molecule is called
• recombinant DNA
• because it contains DNA from 2 different
  sources

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4. Insert vector into cells

• The recombinant DNA must now be inserted into
  cells.
• One method is to use ice cold calcium chloride
  solution

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• The plasmid and bacterial cell to be infected are
  soaked in the solution
• The bacterial cell membrane becomes permeable
• Some plasmids enter

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Not all cells have the plasmid

• To identify the cells with the recombinant
  plasmid a special plasmid is used as the gene
  carrier
• This also has a gene for antibiotic resistance

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• All the infected bacteria are grown on a medium
  containing antibiotic
• Only the bacteria with the plasmid with both
  the antibiotic resistant gene and the gene you
  want will survive

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5. Make the cells multiply

• Remove a sample of the surviving bacterial colony
• Culture it in a large tank a fermenter - with
  nutrients supplied
• Drain off bacteria at intervals

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6. Isolate and purify protein

• Bacterial cell is broken down
• Protein is separated from the cell debris

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Genetically modified foods - advantages

• Better nutritionally
• Better storage time
• Better tasting

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Genetically modified foods - disadvantages

• Health
• 1989 - Some Americans died after using GM food
  supplement
• 1994 - Cancer link to GM growth hormone fed to
  cattle
• 1996 - Brazil nut gene added to soyabeans,
  induced allergic reaction

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Genetic drift

• Supervirus - safe virus can take up harmful
  genes
• Pollen spread - cross pollination possible
• Superweeds - herbicide resistant genes from wheat
  found in other grasses

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Ethics

• Should supermarkets be forced to label GM
  products?
• Should GM companies be liable for genetic
  accidents?
• Should animal and plant genes be blended?

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Moral and Ethical Concerns

• Mutation of transgenic bacteria and viruses -
  new diseases
• Pollen from GM crops could transfer genes to
  other plants - superweeds
• Transgenic animals better competitors for food
  - extinction of other species

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Moral and Ethical Concerns

• Religious objections
• Eugenics
• Screening - discrimination

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The end.

• ..or is it just the beginning!!!