4.4 Biotechnological Tools and Techniques

1
4.4 Biotechnological Tools and Techniques

• Recombinant DNA Gel electrophoresis

2
Recombinant DNA

• Cutting DNA fragments from different sources and
  recombining them together

• Cutting DNA fragments from different sources and
  recombining them together
• 4.4.7 State that, when genes are transferred
  between species, the amino acid sequence of
  polypeptides translated from them is unchanged
  because the genetic code is universal. Obj. 1

• Purpose
• To investigate genetic disorders
• Production of drugs (ie. insulin)

3
What complications do you foresee?

• Consider
• The size of DNA
• Where to cut?
• How to put back together?
• 4.4.8 Outline a basic technique used for gene
  transfer involving plasmids a host cell
  (bacterium, yeast or other cell), restriction
  enzymes (endonucleases) and DNA ligase. Obj. 2

4
1. Restriction Endonucleases

• Also known as restriction enzymes
• Essentially are molecular scissors
• Recognize a specific DNA sequence and cuts the
  strands at a particular position or recognition
  site
• Isolated and purified only from bacteria
• Name reflects which bacteria the enzyme
  originates
• ie. EcoRI ? Escherichia coli, strain R, 1st r.e.
  isolated
• HindII ? Haemophilus influenzae, strain Rd, 2nd
  r.e.

5
1. Restriction Endonucleases Recognition site
Bacteria Restriction Enzyme Recognition Site
Escherichia coli EcoRI 5-GAATTC-3 3-CTTAAG-5
Haemophilus parainfluenzae HindIII 5-AAGCTT-33-TTCGAA-5
Arthrobacter luteus AluI 5-AGCT-33-TCGA-5

• Each restriction endonuclease recognizes its
  own specific recognition site (specific DNA
  sequence)
• Usually 4-8 base pairs long, characterized by a
  complementary palindromic sequence

6
1. Restriction Endonucleases Function

• Scans DNA and binds to its specific recognition
  sequence
• Disrupts the phosphodiester bonds between
  particular nucleotides through a hydrolysis
  reaction
• Hydrogen bonds of the complementary base pairs in
  between the cuts are disrupted
• Result 2 DNA fragments

http//www.scq.ubc.ca/?p249
7
1. Restriction Endonucleases DNA Fragment Ends

• Different DNA fragment ends are produced after
  digestion by different restriction enzymes
• Sticky ends DNA fragment ends with short
  single-stranded overhangs (ie. EcoRI, HindIII)
• Blunt ends DNA fragment ends are fully base
  paired (ie. AluI)

Bacteria Restriction enzyme Recognition site After digestion by restriction enzyme
Escherichia coli EcoRI 5-GAATTC-3 3-CTTAAG-5 5-G AATTC-3 3-CTTAA G-5
Haemophilus parainfluenzae HindIII 5-AAGCTT-33-TTCGAA-5 5-A AGCTT-33-TTCGA A-5
Arthrobacter luteus AluI 5-AGCT-33-TCGA-5 5-AG CT-33-TC GA-5
8
1. Restriction Endonucleases DNA Fragment Ends
(continued)

• http//highered.mcgraw-hill.com/olc/dl/120078/bio3
  7.swf

Restriction site
Animation
Palindrome
Fragment 2
Fragment 1
http//www.bio-rad.com/LifeScience/docs/Official_C
rime_Scene_PowerPoint_Spring_2005_rev_B.ppt
9
How do we control the snips?

• Consider
• What about the organisms own DNA?
• Frequency of recognition sequences within the DNA
  sequence

10
1. Restriction Endonucleases Length of
recognition sites

• http//highered.mcgraw-hill.com/olcweb/cgi/pluginp
  op.cgi?itswf535535/sites/dl/free/0072437316
  /120078/bio38.swfEarly Genetic Engineering
  Experiment

• Longer recognition sites result in lower
  frequency of cuts
• EcoRI ? 5-GAATTC-3 ¼ ¼ ¼ ¼ ¼ ¼
  1/4096
• AluI ? 5-AGCT-3 ¼ ¼ ¼ ¼ 1/256
• Higher frequency of cuts may cut gene into
  several fragments
• Lower frequency of cuts may produce large
  fragments than desired

11
1. Restriction Endonucleases Methylases

• Enzymes that add a methyl group to a nucleotide
  in a recognition site to prevent restriction
  endonuclease from cutting DNA
• Distinguishing between foreign (viral) DNA and
  bacterias own DNA

12
1. Restriction Endonucleases DNA Ligase

• Enzyme that rejoins cut strands of DNA together
  by reforming a phosphodiester bond
• DNA ligase joins sticky ends
• T4 DNA ligase (from T4 bacteriophage) joins blunt
  ends

13
How do we sort out the DNA

• DNA is chopped into many pieces
• How to differentiate one piece from other

14
2. Gel Electrophoresis

• Technique used to separate charged molecules
  based on their size
• 4.4.3 State that gel electrophoresis of DNA is
  used in DNA profiling Obj. 1
• Acts like a molecular sieve

http//www.biotech.iastate.edu/ppt_presentations/h
tml/Fingerprinting/StudentInstruction-gel/images/i
mage08.jpg
http//www.solve.csiro.au/1105/img/sieve-bloke.jpg
15
DNA Profiling

• 4.4.4 Describe the application of DNA profiling
  to determine paternity and also in forensic
  investigations. Obj. 2
• A process of using DNA fragments to identify a
  person, or other organism. The DNA fragments
  have distinct bands separated by spaces and these
  band patterns are so distinct that they can be
  used like fingerprints.

16
2. Gel Electrophoresis DNA Preparation

• Restriction enzymes digest DNA into smaller
  fragments of different lengths
• Different DNA samples are loaded into wells of
  the gel (agarose or polyacrylamide)

http//www.oceanexplorer.noaa.gov/explorations/03b
io/background/molecular/media/gel_plate_600.jpg
17
2. Gel Electrophoresis Attraction Migration

• Negatively charged electrode at the end where
  wells are located
• Positively charged electrode at opposite end
• Negatively charged DNA migrate towards positive
  end due to attraction

18
2. Gel Electrophoresis Rate of Migration

• 4.4.2 State that, in gel electrophoresis,
  fragments of DNA move in an electric field and
  are separated according to their size. Obj. 1
• Shorter/smaller DNA fragments migrate through gel
  faster since they can move through the pores in
  the gel more easily
• Longer/larger DNA fragments migrate through gel
  slower
• Rate of migration 1/log(size)
• Different DNA fragment lengths are separated

A B C D E
A kilobase DNA ladder B uncut plasmid DNA C
single digestion of the plasmid with EcoRI D
single digestion with XhoI E double digestion -
both EcoRI and XhoI.
http//www.answers.com/topic/agarosegel-jpg
19
2. Gel Electrophoresis Visualizing DNA Fragments

• Ethidium bromide is a fluorescent dye that makes
  DNA fragments visible by staining the gel
• DNA fragments can then be isolated and purified

http//www.answers.com/topic/agarosegel-jpg
20
(No Transcript)
21
Paternity Testing
22
4.4.5 Analyse DNA profiles to draw conclusions
about paternity or forensic investigations. Obj.
3
23
Animations

• http//www.sumanasinc.com/webcontent/animations/co
  ntent/gelelectrophoresis.html
• http//learn.genetics.utah.edu/content/labs/gel/
• http//www.dnalc.org/resources/animations/gelelect
  rophoresis.html

24
2. Gel Electrophoresis Proteins too!

• Gel electrophoresis can also be used to separate
  proteins, usually using polyacrylamide gels

http//www.biotechlearn.org.nz/var/biotech/storage
/images/multimedia/images/protein_electrophoresis/
48251-4-eng-GB/protein_electrophoresis_medium.jpg
http//www.bio-link.org/vlab/Graphics/Tools/Protei
nGel2.jpg
25
3. Plasmids

• Small, circular double-stranded DNA that can
  enter and exit bacterial cells
• Lack a protein coat
• Independent of bacterial chromosome
• 1000-200,000 base pairs

26
3. Plasmids Endosymbiosis

• Use host bacterial enzymes and ribosomes to
  replicate and express plasmid DNA
• Carry genes that express proteins to protect
  bacteria against antibiotics and heavy metals

27
3. Plasmids

• Foreign genes (ie. insulin) can be inserted into
  plasmids, so bacteria can express gene and make
  its respective protein
• Higher copy number of plasmids (number of
  individual plasmids) in bacteria
• results in larger number of gene copies, thus
  more of its respective protein is synthesized

28
3. Plasmids

• Restriction endonucleases splice foreign genes
  into plasmids
• DNA ligase reforms phosphodiester bond between
  the fragments, resulting in recombinant DNA

• http//www.learner.org/courses/biology/archive/ani
  mations/hires/a_gmo1_h.html

http//www.accessexcellence.org/RC/VL/GG/inserting
.html
29
4. Transformation

• Introduction of foreign DNA (usually a plasmid)
  into a bacterium
• Plasmids can be used as a vector (vehicle that
  DNA can be introduced to host cells) to carry a
  specific gene into a host cell

http//www.bio.davidson.edu/Courses/Molbio/MolStud
ents/spring2003/Siegenthaler/fig2.gif
30
4. Transformation Competence

• Competent cell - Bacterium that readily takes up
  foreign DNA (ie. able to undergo transformation)
• Most cells are not naturally competent, but can
  be chemically induced to become competent

• Calcium ion in calcium chloride stabilizes
  negatively charged phosphates on bacterial
  membrane

31
4. Transformation Competence
32
4.4.9 State two examples of the current uses of
genetically modified crops or animals. Obj. 1

• The transfer of a gene for factor IX which is a
  blood clotting factor, from humans to sheep so
  that this factor is produced in the sheeps milk.
  
• The transfer of a gene that gives resistance to
  the herbicide glyphosate from bacterium to crops
  so that the crop plants can be sprayed with the
  herbicide and not be affected by it. 

33

• Issues Surrounding Genetically Modified (GM)
  Products
• by Subhuti Dharmananda, Ph.D., Director,
  Institute for Traditional Medicine, Portland,
  Oregon

34
Meet the Super Cow

• http//www.youtube.com/watch?vNmkj5gq1cQU

35
4.4.10 Discuss the potential benefits and
possible harmful effects of one example of
genetic modification. Obj. 3

• Benefits
• Crops
• Enhanced taste and quality, Reduced maturation
  time, Increased nutrients, yields, and stress
  tolerance, Improved resistance to disease, pests,
  and herbicides, New products and growing
  techniques
• Animals
• Better yields of meat, eggs, and milk
• Improved animal health and diagnostic methods
• Increased resistance, productivity, hardiness,
  and feed efficiency
• Environment
• "Friendly" bioherbicides and bioinsecticides
• Conservation of soil, water, and energy
• Bioprocessing for forestry products
• Better natural waste management
• More efficient processing
• Society
• Increased food security for growing populations

36
Controversies

• Safety
• Potential human health impact allergens,
  transfer of antibiotic resistance, unknown
  effects.
• Potential environmental impact unintended
  transfer of transgenes through cross-pollination,
  unknown effects on other organisms (e.g., soil
  microbes), and loss of flora and fauna
  biodiversity
• Access and Intellectual Property
• Domination of world food production by a few
  companies
• Increasing dependence on industrialized nations
  by developing countries
• Biopiracy-foreign 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 (e.g., United
  States)
• Mixing GM crops with non-GM confounds labeling
  attempts
• Society
• New advances may be skewed to interests of rich
  countries
• Dharmananda, S. Issues Surrounding Genetically
  Modified (GM) Products. Online
  http//www.google.ca/imgres?imgurlhttp//www.itmo
  nline.org/image/gmo1b.jpgimgrefurlhttp//www.itm
  online.org/arts/gmo.htmusg__y5yFCjq561X8N0vAy5g8
  nid85NMh453w400sz32hlenstart5zoom1tb
  nidKqfgq2YL9RdYEMtbnh127tbnw112eik9TfTpasG
  sfL0QG8hdCuBwprev/search3Fq3Dgmo26um3D126hl
  3Den26safe3Dactive26sa3DN26gbv3D226tbm3Di
  schum1itbs1