The use of living things, biological systems and processes for the benefit of humans.

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• The use of living things, biological systems and
  processes for the benefit of humans.

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1. Manipulating DNA

• EL To learn how to cut and paste DNA

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Tools for manipulating DNA
1. CUTTING DNA into fragments using restriction enzymes these molecules cut at specific DNA sequences and are only found in prokaryotic organisms
2. PASTING Pasting DNA fragments together using enzymes called ligases. We can join fragments of DNA to make what is called recombinant DNA. DNA ligases found in many species including humans.
3. COPYING Making many copies of DNA (amplification) using DNA polymerases in the Polymerase Chain Reaction (PCR) technique. All organisms contain DNA polymerases as they all need to copy their DNA.
4. TRANSFERRING DNA into cells using vectors such as plasmids. This technique is called a transformation in prokaryotic cells.
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1. Cutting DNA

• Restriction enzymes (molecular scissors) are
  found in prokaryotic organisms

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Specificity

• Restriction enzymes are specific
• The DNA and the enzyme need to be mixed together
  and incubated at a temperature that will result
  in maximum activity of the enzyme.
• Each restriction enzyme will only cut the DNA at
  a specific sequence of A, G, T and Cs. We call
  this place a recognition site.

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Different restriction enzymes recognise specific
recognition sites.
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Cutting Specificity

• When DNA is cut with a restriction enzyme the
  resulting fragments are left with either a short
  overhang of single stranded DNA called a sticky
  end or no overhanging DNA which is called a blunt
  end (snake demo)

EcoRI leaves sticky ends GAATTC CTTAAG
HpaI leaves blunt ends
GTT AAC
GTTAAC
CAA TTG
CAA TTG
- site where enzymes cuts through the
sugar phosphate backbone of the DNA strand.
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Whats in a name!

• Restriction enzymes are named after the organism
  from which they were isolated.
• E.g. Escherichia coli
• EcoRI
• The Roman number indicates the order of discovery
• If another letter is placed in front of the Roman
  number it signifies a particular strain of the
  bacterium. R resistance

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Restriction Enzyme Restriction Site Overhang Type
EcoRI E genus Escherichia co species coli R strain RY13 I first endonuclease isolated GAATTC CTTAAG STICKY
BamHI B genus Bacillus am species amyloliquefaciens H strain H I first endonuclease isolated GGATCC CCTAGG STICKY
HindIII H genus Haemophilus in species influenzae d strain Rd I third endonuclease isolated AAGCTT TTCGAA STICKY
HpaI H genus Haemophilus pa species parainfluenzae I first endonuclease isolated GTTAAC CAATTG BLUNT
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Fragments are sorted by Gel Electrophoresis

• This technique is used to separate out fragments,
  obtained by a restriction digest, of DNA
  according to their size (length in base pairs).
• DNA fragments are separated into bands containing
  fragments of the same length by electrical
  separation in a gel matrix.
• DNA molecules migrate to the positive electrode,
  when an electric field is applied to the gel
  matrix, as they are negatively charged.
• This technique is used to isolate DNA fragments
  containing genes which are subsequently used to
  make recombinant DNA.

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Fragments are sorted by Gel Electrophoresis
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2. Pasting

• When two samples of DNA are combined using DNA
  ligases.
• Any 2 DNA strands can be joined that have
  complementary exposed nucleotides (i.e. cut with
  same restriction enzyme).

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Activities

• Watch DNAi animations
• Complete activity 9.1 Gel electrophoresis
  analysis 9.2 Detecting traits in families.

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Reflection

• Summarise in your own words restriction
  enzymes, cutting, pasting and gel electrophoresis
• What learning was new today?
• What learning was revision or built on what I
  already know?
• What did I find most challenging and what
  strategies will I put in place to help me?
• What percentage of the class did I spend on task
  and how can I improve this if needed

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2. Manipulating DNA

• EL To learn how to copy and transfer DNA.

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3. Copying (Amplification) of DNA using the
Polymerase Chain Reaction

• The 300 million dollar man.

• I was working for Cetus, making oligonucleotides
  (primers). They were heady times. Biotechnology
  was in flower and one spring night while the
  California buckeyes were also in flower I came
  across the polymerase chain reaction. It was the
  first day of the rest of my life.
• Kary Mullis 1972

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Why PCR?

• To amplify a small amount of DNA into an
  analysable quantity
• E.g. crime scene, fossils etc

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PCR Tools

• Taq DNA Polymerase is an enzyme that works well
  at 72C.

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PCR Tools

• Primers
• Synthetic short segments of DNA up to 25
  nucleotides long.
• Probe for a specific sequence or gene along a
  strand of DNA.
• Hybridise with a sequence of bases on the
  template DNA through complementary base pairing.
• Indicate to Taq DNA polymerase where to start
  building the complementary strand by extending
  the primer.

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Find the starting point for copying STR regions

• Select your primer
• Start region Sequence to be copied by extending
  the primer.

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Thermocycling machine
Step 1. Denaturation

• Step 1 Denaturing the DNA 2 minutes

T A C C G T A A A T G C C
A T T
Step 1 92C

• At this temperature the hydrogen bonds are broken
  resulting in two single strands of DNA.

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Step 2. Attachment of Primers
T A C C G T A A
A T G
T A A
A T G C C A T T
Step 2 55C

• Step 2 Primer annealing 2 minutes
• The temperature is lowered to allow the primers
  to bind (anneal) to their complementary bases on
  each of the single strands of DNA.

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Step 3 Extension
T A C C G T A A
A T G G C
Step 3 72C
G G T A A
A T G C C A T T

• Step 3 DNA synthesis 1 minute
• Taq DNA polymerase extends the DNA strand from
  the primers using the base pairing rule.

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And you can repeat the three step cycle over and
over!
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PCR song

• http//www.youtube.com/watch?vdD3faDLEvmYfeature
  related

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4. Transferring
Fluorescent jellyfish

• Because DNA is the same in all organisms, we
  should be able to take a piece of DNA from one
  organism and put it into another organism.
• You can change the way an organism looks or
  behaves!
• This process of taking DNA from one organism and
  putting into another is called transformation.

Plasmid
Jellyfish and plasmid DNA is cut with the same
restriction enzyme.
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Vectors

• Gene inserted into a vector that will carry the
  gene into the desired organism.
• Common vectors are
• Viral vectors (eg. Adenovirus and retorovirus)
  must have disease symptom genes removed first!
• Liposome vectors small circular molecules
  surrounded by phospholipid bilayer
• Plasmid vectors small circular piece of
  bacterial DNA. Plasmids are used as vectors in
  bacterial transformations.

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Plasmids are not naturally attracted to bacteria!
Bacterium
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Transformation of Bacteria with a Recombinant DNA
Plasmid

• Making the bacteria more attractive to plasmids
  
• Plasmids are
• now attracted
• to the bacteria

Bacterium
CaCl2 solution
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The Transformation

• Now give the bacteria some food and the right
  temperature to reproduce.
• Any bacteria with the plasmid inside will start
  making the jelly protein, that results in
  fluorescence.

HEAT SHOCK
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Activity

• In pairs, complete Activity 12.2 finding a gene
• Quick check qu 1-3 (pg 426), 4-6 (pg 431)
• Ch 12 review qu 3, 4, 5, 6, 7, 8, 12

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Reflection

• Summarise in your own words copying (include
  PCR) and transferring
• What learning was new today?
• What learning was revision or built on what I
  already know?
• What did I find most challenging and what
  strategies will I put in place to help me?
• What percentage of the class did I spend on task
  and how can I improve this if needed

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3. Applications of DNA manipulation

• EL To explore the uses of DNA manipulation.

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Gene Sequencing

• Gene sequencing is identifying the nucleotide
  order in a segment of RNA or DNA.
• A G G A C T C A T G G A G A A G A A C T T T . . .
• Our genome has been sequenced. We have
  3,100,000,000 base pairs, what a big book!

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Gene Cloning

• Making identical copies of sequences of DNA that
  code for proteins using plasmids
• extract plasmid from bacteria
• Cut plasmid DNA and DNA of the gene to be
  inserted with same restriction enzyme
• Paste 2 pieces of DNA using DNA ligase to create
  a recombinant plasmid.
• Add recombinant plasmid to bacterial culture,
  where some are taken up and replicate (called
  transformation)
• Isolate and analyse bacteria containing
  recombinant plasmids.
• PRACTICAL APPLICATION Production of human growth
  hormone

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DNA Profiling

• Compares base sequence of 2 or more individuals
• Short tandem repeats (STRs) and variable
  nucleotide tandem repeats (VNTRs) non-coding
  sections of DNA repeated many times between genes
• E.g. GAGAGAGAGAGAGA
• There are more than 10,000 STR loci in one set
  of human chromosomes!

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DNA Profiling

• The repeat is present in all members of the
  population, but the number of repeats varies
  among individuals and is inherited.
• DNA profiling allows us to view these patterns in
  our DNA.
• Uses PCR and gel electrophoresis smaller
  fragments will migrate further on the gel.

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DNA Profiling
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DNA Profiling

• Loci of STR regions found to vary from person to
  person with a high frequency
• 13 are used in America, but only 9 are used in
  Australia why?

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Activities

• Genetic Engineering A model (Biol The Common
  Threads, pg 175)
• DNA fingerprinting (Biol The Common Threads, pg
  179)
• Quick check qu 7-10 (pg 433), 11-14 (pg 437),
  15-17 (pg 443), 18-21 (pg 448), 22-24 (pg 455),
  2526 (pg 457)
• Ch 12 ch review qu 9, 10, 11 (pg 461)

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Reflection

• Summarise in your own words gene sequeencing,
  cloning and DNA profiling
• What learning was new today?
• What learning was revision or built on what I
  already know?
• What did I find most challenging and what
  strategies will I put in place to help me?
• What percentage of the class did I spend on task
  and how can I improve this if needed