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TECHNIQUES USE IN
GENETIC ENGINEERING
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DNA EXTRACTION
ELECTROFORESIS
HYBRIDITATION
PCR
SEQUENCING
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Analysis of DNA
• gel electrophoresis- separates DNA
fragments based on size
• nucleic acid hybridization & probes –
probes base pair with complementary
sequences; used to detect specific
sequences
• DNA Sequencing – reading the sequence
of nucleotides in a stretch of DNA
• Polymerase Chain Reaction – way to
amplify DNA
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DNA EXTRACTION
There are three basic and two optional steps in a
DNA extraction:
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Breaking the cells open, commonly referred to as
cell disruption or cell lysis, to expose the DNA
within. This is commonly achieved by chemical
and physical methods-blending, grinding or
sonicating the sample.
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DNA EXTRACTION
2
Removing membrane lipids by adding a detergent
or surfactants.
3
Removing proteins by adding a protease (optional
but almost always done).
4
Removing RNA by adding an RNase (often done).
5
Precipitating the DNA with an alcohol — usually
ice-cold ethanol or isopropanol. Since DNA is
insoluble in these alcohols, it will aggregate
together, giving a pellet upon centrifugation. This 5
step also removes alcohol-soluble salt.
DNA EXTRACTION
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DNA EXTRACTION
ELECTROFORESIS
HYBRIDITATION
PCR
SEQUENCING
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Gel Electrophoresis
•
Electrophoresis - the migration of charged
molecules in an electric field though a solution or
solid support
•
Various types – defined by support used
1.
Paper – amino acids, small peptides
2. Polyacrylamide – Proteins, small DNA/RNA
(<500bp)
3. Agarose – DNA/RNA
•
Good preparative and analytical method
Gel Electrophoresis
• Separation of charged molecules in
an electric field.
• Nucleic acids have 1 charged
phosphate (- charge) per nucleotide.
means constant chare to mass ratio.
Separation based (mostly) on length:
longer molecules move slower.
• Done in a gel matrix to stabilize:
agarose or acrylamide.
• average run: 100 Volts across a 10 cm
gel, run for 2 hours.
• Stain with ethidium bromide:
intercalates between DNA bases and
fluoresces orange.
• Run alongside standards of known
sizes to get lengths
Gel
electrophoresis
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Gel Electrophoresis
Gel Electrophoresis
•
Gel electrophoresis uses a cross-linked
polymers (agarose) that contain various
pores.
•
Pores allow molecular sieving, where
molecules e.g. DNA, can be separated based
upon there mobility through the gel.
Gel Electrophoresis
Mobility = Charge + Molecular Dimensions
• Charge per nucleic acid is
constant
•This means separation is based
upon length of the DNA molecules
and this is how we can separate
and identify DNA molecules.
Gel Electrophoresis
• Linear DNA has a linear relationship to
distance migration.
• If add molecular markers of known mass
can calculate mass of our fragment by
plotting a linear plot.
Gel Electrophoresis
•
1.
2.
3.
Other factors determining mobilityPolymer concentration e.g. Agarose
Conformation of DNA
Electrophoresis
Gel Electrophoresis
•
1.
2.
3.
Detection
Dye e.g. ethidium bromide
Audioradiography 32P,
Blotting (see later)
•
1.
Uses
Analytical- Can determine size of DNA
fragment,
Preparative – Can identify a specific fragment
based on size
2.
DNA EXTRACTION
ELECTROFORESIS
HYBRIDITATION
PCR
SEQUENCING
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hydridization
• The idea is that if DNA is made single
stranded (melted), it will pair up with
another DNA (or RNA) with the
complementary sequence. If one of the
DNA molecules is labeled, you can detect
the hybridization.
• Basic applications:
• Southern blot: DNA digested by a restriction
enzyme then separated on an electrophoresis gel
• Northern blot: uses RNA on the gel instead of
DNA
• in situ hybridization: probing a tissue
• colony hybridization: detection of clones
• microarrays
hydridization
Applications
• The main use of this technique is to identity any changes in
DNA sequencing or genes expressed, e.g. comparing genes
expressed by a diseased cell to genes expressed by an
healthy cell.
• Other uses include- Testing for hereditary disease,
Evolutionary history of species, Screening e.g.food
supply
• Applications to synthetic biology
- identification of various parts in natural organisms,
-?more?
Hybridization Process
• All the DNA must be single stranded (melt
at high temp or with NaOH). Occurs in a
high salt solution at say 60oC.
Complementary DNAs find each other and
stick. Need to wash off non-specific binding.
• Stringency: how perfectly do the DNA
strands have to match in order to stick
together? Less than perfect matches will
occur at lower stringency (e.g. between
species). Increase stringency by increasing
temp and decreasing salt concentration.
• Rate of hybridization depends on DNA
concentration and time (Cot), as well as GC
content and DNA strand length.
• Autoradiography. Put the labeled DNA
next to X-ray film; the radiation fogs the
film.
Labeling
• Several methods. One is random
primers labeling:
• use 32P-labeled dNTPs
• short random oligonucleotides as
primers (made synthetically)
• single stranded DNA template
(made by melting double
stranded DNA by boiling it)
• DNA polymerase copies the DNA
template, making a new strand
that incorporates the label.
• Can also label RNA (sometimes
called riboprobes), use nonradioactive labels (often a small
molecule that labeled antibodies
bind to, or a fluorescent tag), use
other labeling methods.
hydridization
•
•
1.
2.
3.
Using specific probes that are labelled specific
sequences of DNA can be identified.
There are three main hybridization techniques
which vary in the sample blotted and the
probes used;
Northern Blot-Transfer of an RNA sample
separated and identified using DNA or RNA
probes.
Southern Blot-Transfer of an DNA sample
separated and identified using DNA or RNA
probes.
Western Blot- Transfer of an Protein sample
separated and identified typically using an
antibody.
hydridization
• Blotting – Transfer of DNA, RNA or Proteins, typicall
from a electrophoresis gel to a membrane e.g.
nitrocellulose. This membrane can then be subject to
further techniques such as hybridization.
• Hybridization – Process where two complementary
single strands of nucleic acid (DNA or RNA) form a
double helix.
Southern blot
hydridization
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In situ hybridization
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DNA EXTRACTION
ELECTROFORESIS
HYBRIDITATION
PCR
SEQUENCING
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Polymerase Chain Reaction (PCR)
•
A method for amplifying specific DNA
sequences.
•
Components required:
- Target sequence
- A pair of primers
- dNTPs (ATGC)
- DNA polymerase
Polymerase Chain Reaction (PCR)
•
Five noteworthy features of PCR:
1) The sequence of the target need
not be known.
2) The target can be much larger
than the primers (>10 kb).
3) Primers do not have to perfectly
match flanking sequences.
4) Stringency can be controlled by
temperature and salt (MgCl2).
5) PCR is very sensitive.
Polymerase Chain Reaction (PCR)
• One PCR cycle involves three steps:
- Strand separation (95ºC)
- Hybridization of primers (54ºC)
- DNA synthesis (72ºC)
• After n cycles, the sequence is
amplified 2n-fold.
PCR
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Polymerase Chain Reaction (PCR)
• Based on DNA polymerase creating a second strand of
DNA.
• Needs template DNA and two primers that flank the region to
be amplified. Primers are short (generally 18-30 bases) DNA
oligonucleotides complementary to the ends of the region being
amplified.
• DNA polymerase adds new bases to the 3' ends of the primers to
create the new second strand.
• go from 1 DNA to 2, then 4, 8, etc: exponential growth of DNA
from this region
• A key element in PCR is a special form of DNA polymerase from
Thermus aquaticus, a bacterium that lives in nearly boiling
water in the Yellowstone National Park hot springs. This
enzyme, Taq polymerase, can withstand the temperature cycle
of PCR, which would kill DNA polymerase from E. coli.
• advantages:
• rapid, sensitive, lots of useful variations, robust (works even with
partly degraded DNA)
• disadvantages:
• Only short regions (up to 2 kbp) can be amplified.
• limited amount of product made
Polymerase Chain Reaction (PCR)
PCR Cycle
• PCR is based on a cycle of 3 steps
that occur at different
temperatures. Each cycle doubles
the number of DNA molecules: 2535 cycles produces enough DNA to
see on an electrophoresis gel. Each
step takes about 1 minute to
complete.
•
1. Denaturation: make the
DNA single stranded by heating to
94oC
•
2. Annealing: hybridize the
primers to the single strands.
Temperature varies with primer,
around 50oC
•
3. Extension: build the
second strands with DNA
polymerase and dNTPs: 72oC.
Other PCR Images
DNA Amplification in PCR
• original DNA: very long molecules
with neither end well defined.
Number stays constant in the
PCR reaction: no new ones are
made.
• initial PCR product made from
original DNA: has one end
defined by the primer, but the
other end is not well defined.
Copy number grows linearly.
• all other PCR products have 2
ends defined by the primers, so
they have a constant length and
can be easily detected by
electrophoresis. Copy number
grows exponentially.
DNA EXTRACTION
ELECTROFORESIS
HYBRIDITATION
PCR
SEQUENCING
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DNA Sequencing
• DNA Sequencing – Determining the order of nucleotides
in a DNA molecule
• Key technique as it can give us information about a DNA
molecule, e.g. location and order of genes, restriction sites.
• In addition, for recombinant DNA gives verification of
gene cloning experiments.
• 2 possible use’s for project – Identify sequence of new part,
- Checking recombinant DNA.
Sanger
technique
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DNA Sequencing
Deoxyribonucleotide acid
This is essentially the monomer of
DNA. Polymerization of nucleotides
occurs by condensation reaction of a
5’ phosphate to a 3’ hydroxyl group
Dideoxyribonucleotide acid
There is no 3’hydroxyl group to
allow polymerization.
DNA Sequencing
THANK YOU
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