Download BCM301 Food Biotechnology

BCM302 Food and Beverage
Biotechnology
Topic 3:
Basic Principles of Recombinant
DNA technology
Learning objectives
1.
2.
3.
4.
5.
6.
Know the function of restriction endonucleases, how they work to cut
DNA, and why they are important in biotechnology. Compare blunt
ends with sticky ends.
Know the mechanism by which electrophoresis separates pieces of
DNA.
List and know the steps of DNA cloning.
Know how vectors are used to transform bacteria, and know the
methods of selecting for successfully transformed bacteria. Compare
the types of vectors in terms of the sizes of DNA that can be carried
by the vector into the bacteria.
List the types of vectors that can be used to transform yeast
mammalian cells and plants, and why they are effective in those
organisms.
List the methods of transformation of cells.
Learning objectives
7. Compare genomic libraries, cDNA libraries, and expression libraries in terms of
how they are constructed, what the libraries are looking for, and how they are
screened.
8. List the various types of reporter genes used in research.
9. Compare Northern and Southern blot hybridization in terms of how they are
constructed and what each type of hybridization is looking for.
10. Know the function of PCR, the steps of PCR, and what PCR allows researchers to
accomplish.
11. Compare the two methods of DNA sequencing: the chemical method and the
Sanger method, and know which method is more widely used. How does automation
impact DNA sequencing?
12. List and define the various methods of analyzing proteins. Are any of these
methods similar to DNA methods?
13. Know the types of microarrays, and how DNA and protein microarrays work.
14. List the applications of recombinant DNA technology.
Recombinant DNA Technology:
Promise and Controversy
• What is DNA cloning?
• Applications:
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–
–
–
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New medical diagnostics and treatments
Better vaccines
Stress-resistant crops
More nutritious food
Healthier livestock
Cleaner environment
Recombinant DNA Technology:
Promise and Controversy
• Criticisms:
– Where to draw the line (human cloning, organs
for donation, selecting characteristics in
engineered children)
– Effect on health, hidden dangers,
environmental pollution (eg escaping genes and
organisms)
– Tampering with the “natural world”
• Solutions?:
– Open communication
Cutting and Joining DNA
• Recombinant DNA: When two pieces of
DNA are joined together to form a new
DNA molecule
• Cloning: Insertion of DNA molecules in
bacteria so that many identical copies of
the DNA are made
• Gene expression: DNA can be transcribed
and protein can be translated within cell
Restriction enzymes
• Also called restriction endonucleases
• Cut DNA at “restriction sites or
“restriction sequences”
• Cut across the sugar-phosphate
backbone of DNA
Restriction enzymes
• Palandromic, Four-six bases in length
Restriction enzymes
• Derived from bacteria
• Natural role: defence against
bacteriophage
• Bacterial DNA protected by methyl
groups on adenine or cytosine
Restriction enzymes
• Blunt ends: enzyme cuts directly
across the two strands
• Sticky ends: enzyme cuts strands
in different places, leaving a short
single stranded piece of DNA
hanging over the end of the
molecule
Restriction enzymes
• Blunt vs Sticky
Separating restriction fragments
• Digestion of DNA with Restriction enzymes
may result in fragments of different sizes
which need to be separated
• DNA passed through a “molecular sieve” to
separate molecules of different sizes
Agarose gel
• Agarose: polysaccharide derived from
seaweed
• Powder mixed with buffer, heated and
forms a solid gel when cooled
• “Wells” formed in gel during cooling for
sample loading
Electrophoeresis
• Electric charge applied to allow DNA to migrate (towards
+ve electrode)
• DNA slightly negative due to sugar-phosphate backbone
• Large molecules migrate slower
• Small molecules migrate faster
• Size of pores determined by Agarose concentration
– Lower % = larger pores = better separation for larger molecules
– Higher % = smaller pores = better separation for smaller molecules
Visualizing DNA
• Ethidium bromide: binds to DNA and
fluoresces when exposed to UV light
• Size of DNA fragments determined by
comparing with fragments of know sizes
• DNA fragments migrate at a rate that is
inversely proportional to the logarithm of
the size of the fragment (in base pairs)
Agarose gel Electrophoresis
Acrylamide gel electrophoreis
• Gel formed using
polyacrylamide
• Vertical
• Used for smaller
molecules (eg DNA
sequencing)
DNA cloning
• Isolation of DNA
• Ligation of the DNA to a vector
• Transformation of a host cell with
the recombinant DNA
• Selection of host cells containing the
DNA
Isolation of DNA
• Cleaved from a larger piece of DNA
• Generated using the Polymerase Chain
Reaction (see later)
Ligation
• DNA ligated to a
cloning vector
• Enzyme: Ligase
• Requires compatible
ends
Cloning Vectors
• Transport molecule so DNA can be
replicated in cell:
• Features:
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–
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Origin of replication (Ori)
Small
Multiple cloning site (MCS) with unique RE sites
Selectable marker (to determine which bacteria
contain the vectors with the inserted DNA)
Bacterial vectors (Plasmids)
• Extrachromosomal pieces of DNA not
necessarily needed by bacteria
• Natural roles eg antibiotic resistance
pigment production
• Engineered to accept DNA fragments up to
10 kilobases in length
• High copy number and low copy number
Plasmid: pBR322
• Contains unique
REs
• Antibiotic
resistance (to
determine which
bacteria contain
vectors)
Plasmid: pUC18/19
Alpha complementation
• lacZ gene codes for betagalactosidase
• Beta-galactosidase breaks down X-gal
to produce a blue product
• If molecule cloned into MCS (in lacZ
gene) X-gal will not be broken down
and colony will be white
Colony selection
Other vectors
• Bacteriophage
– virus that infects bacteria
• Cosmids
– Has aspects of both plasmid and bacteriophage
• Yeast Artificial Chromosome (YAC)
– Contains centromeere, telomere, autonomously replicating
sequence (ARS), selectable marker gene
• Bacterial Artificial Chromosomes (BAC)
– Created using a small plasmid with a F (fertility) factor
that allows the vector to accommodate larger pieces of
DNA (up to 25% of the size of the bacterial
Chromosomes)
Plant Cloning Vector
• Tumor inducing
(Ti) plasmid
• Replicates in
both E.coli. and
Agrobacterium
• Facilitates plant
transformation
Cell transformation
• Transformation: insertion of DNA into a
cell
• Methods:
– Calcium Chloride/heatshock (Bacteria)
– Electroporation (Bacteria, mammalian,
protoplasts)
– Microinjection (Mammalian)
– Biolistics (Plant)
Plant Transformation
• DNA coated onto
small particles
(eg gold or
tungsten)
• Accelerated using
a particle gun
DNA Libraries
• Used to help map genomes
• Can be screened to find specific
genes
• Different types:
– Genomic library
– cDNA library
Genomic Library
• Digest genomic
DNA and plasmid
• Ligate DNA to
vector
• Transform
bacteria with
recombinant
vectors
• Screen
cDNA Library
• A DNA library constructed from
cDNA
• cDNA (complementary DNA): a DNA
copy of messenger RNA (mRNA)
cDNA synthesis
• Pre-mRNA is
transcribed by cell
• Introns are
removed
• A single stranded
cDNA copy is made
using reverse
transcriptase
cDNA synthesis (con’t)
• mRNA is
degraded
• Second DNA
strand
synthesised
using DNA
polymerase
cDNA Library
• cDNA can be derived from RNA
isolated from specific organism,
tissue or treatment
• cDNA ligated to vector as per
genomic DNA
Screening libraries
• Bacterial colonies can be screened
for the presences of specific:
– DNA (southern hybridisation)
– RNA (northern hybridisation)
– Proteins (western hybridisations)
Colony Hybridisaton
• DNA transferred to membrane
• Membrane probed with complementary DNA
Probe hybridisation
• DNA probe with complementary
sequence will bind to DNA from
colony
Reporter genes
• Connected to
gene of interest
to study
expression
pattern
• Eg: Luciferase,
GFP, GUS
Southern Hybridisation
• Invented by Edward Southern in
the mid-1970s
• Allows the detection of a DNA
fragment in a large population of
molecules
Southern Hybridisation
• Steps:
– DNA fragments can be separated on a gel and
denatured
– Fragments transferred to a nylon or nitrocellulose
filter
– Radioactive DNA probes hybridised to membrane
– Position of radioactivity (detected by
autoradiography) indicates position of DNA of
interest
Southern Hybridisation
Southern Hybridisation
Southern Hybridisation
Northern Hybridisation
• Similar to Southern hybridisation
except RNA used instead of DNA
• Used to assess levels of gene
expression
Western hybridisation
• Similar to Southern and Northern
Hybridisation except it involves
detection of proteins with antibodies
Polymerase chain reaction (PCR)
• Amplification of specific pieces of
DNA
• Three steps (cycles) repeated 25-40
times:
– Denaturation (Double stranded melted)
– Annealing (primers bind to specific seq.)
– DNA synthesis (DNA replicated)
Polymerase chain reaction
Polymerase chain reaction
• Primers:
– Short pieces of single stranded DNA (oligonucleotides)
complementary to sequence to be amplified
• DNA polymerase:
– Enzyme that can
synthesise DNA suing a
DNA template
– Requires double stranded
molecule to initiate
synthesis
Steps in PCR
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Applications of PCR
• Isolate specific DNA for further
analysis
• Generate DNA fingerprint to
determine genetic relationships
• DNA sequencing
DNA sequencing
• Two types:
– Chemical method: Selective degradation
– Dideoxy method: generates fragments
of different sized during replication by
interrupting synthesis
DNA sequencing
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
DNA sequencing
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Protein Gel Electrophoresis
• Two types of gels:
– One dimension
– Two dimension
1-D Gel electrophoresis
• Vertical polyacrylamide gels
• Contains sodium dodecyl sulphate
(SDS) to interrupt hydrogen
bonding
• Proteins run based on size
• Visualised by staining (coomassie
blue) or radiolabeling
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
2-D gel electrophoresis
• First dimension: proteins
separated based on charge
• Second dimension: proteins
separated based on size
• Visualised by staining (silver
stain) or radiolabelling
• Applications:
– Analysing whole cell protein
extracts
– Assessing different forms (eg
glycosylated) of a protein
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Protein engineering
• Protein sequence changed by altering
the DNA sequence
Applications of Protein engineering
• Applications
– Resistance to degradation, pH change,
temp., oxidation
– Enhance activity
– Change substrate specificity
– Increase nutritional value
Protein sequencing
• Edman degradation method
• Mass spectrometry
DNA microarray technology
• Whole genome analysis
• Can compare all genes expressed in
diff. tissues/diff. Conditions
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Microarray procedure
• Step 1: Prepare array
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Microarray procedure
• Step 2: Preparing
and hybridizing
labelled target
cDNA
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Microarray procedure
• Step 3:
Analysis
RNA interference technology
Additional resources
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www.protocol-online.org
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www.biology.arizona.edu/molecular_bio/problem_sets/Recombinant_DNA_Technology/recombinant_dna.html
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Gel Electrophoresis of DNA and RNA—in-depth descriptions of the concepts behind gel electrophoresis of nucleic acids.
us.expasy.org/ch2d/protocols/
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Virtual Lab: Agarose Electrophoresis—s Java applet simulation of DNA gel electrophoresis that allows you to start and end
electrophoresis, as well as see a gel as it might appear under ultraviolet light.
arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/
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Gel Electrophoresis Simulator—a Java applet that allows you to enter the sizes of DNA pieces and see where they may travel on an
electrophoresis gel.
arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/virgel.html
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DNA Microarray Methodology Animation—an animated description of DNA microarray experiments.
webphysics.davidson.edu/applets/biogel/biogel.html
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Recombinant DNA—descriptions of DNA cloning techniques, hybridization techniques, PCR, and DNA fingerprinting technology. This
site also contains problems that test your knowledge of these techniques.
www.bio.davidson.edu/courses/genomics/chip/chip.html
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Recombinant DNA Technology Problem Set—questions dealing with PCR, recombinant DNA technology, and Southern blot analysis, along
with applications of these techniques.
web.mit.edu/esgbio/www/rdna/rdnadir.html
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Protocol Online—a searchable list of laboratory protocols in molecular biology, cell biology, biochemistry, and proteomics.
Technical Information on 2-D PAGE—a web site dealing with the theories behind 2-dimensional gel electrophoresis of proteins (2-D
PAGE).
http://www.cc.ndsu.nodak.edu/instruct/mcclean/plsc431/cloning/clone3.htm
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Cloning and Molecular Analysis of Genes—a description of the types of vectors used in DNA cloning.
Additional resources
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www.dnalc.org/shockwave/southan.html
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www.dnalc.org/shockwave/pcranwhole.html
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BioMath Calculators—a Web-based series of calculators for common calculations in molecular biology, such as the melting temperature
of short pieces of DNA called oligos, conversion of absorbance to concentration of DNA in a sample, and dilution calculations. Hosted
by Promega Corporation.
homepages.gac.edu/~cellab/index-1.html
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BioTechniques—a monthly magazine devoted to covering new developments regarding techniques in biotechnology.
www.promega.com/biomath/default.htm
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Green Fluorescent Protein (GFP) Links for Teachers—hosted by the American Society for Cell Biology, this Web site provides
information regarding one of the more popular reporter proteins used in biotechnology research.
www.biotechniques.com
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Clone Library Screening—a short description on how DNA and genomic libraries are screened.
www.ascb.org/teachers/green.html
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DNALC: PCR Animation—an animated tutorial of the theory and steps of the Polymerase Chain Reaction (PCR), along with an example of
PCR in action.
www.ndsu.nodak.edu/instruct/mcclean/plsc431/cloning/clone5.htm
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DNALC: Southern Blotting—an animated tutorial of the technique of Southern blot analysis.
Cell Biology Laboratory Manual—a collection of laboratory protocols in cell biology, along with a list of links to other Web sites related
to cell and molecular biology.
www.carolina.com/biotech/Default.asp
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Carolina Biological: Biotechnology and Genetics—hosted by Carolina Biological Supply Company, this Web site provides information and
suggestions for presenting various topics in biotechnology to students. Links to other Web sites, as well as video clips are also
available.