DNA Isolation, PCR &

Sequencing
Dr. dr. Mgs. Irsan Saleh, M.Biomed
Bagian Farmakologi FK UnsriPalembang
E-mail: irsan_saleh_hasani@yahoo.com

DNA Isolation
Blood Samples
DNA extraction
PCR amplification of the gene of interest
Analysis
Chromosome 17
RFLP
Sequencing
Analysis Analysis
Micropipettors come
in a range of sizes.
They have
disposable tips that
hold tiny amounts of
required reagents.
Lysate Fractionation by Density Gradients
When a solution of cesium chloride is centrifuged at extremely high
speed, the cesium ions tend to sink slightly, forming a density
gradient along the tube. Any other substance in the tube will float at
the point where its density matches that of the gradient.
--> yields VERY pure DNA, but it takes very long time to perform
PCR Agarose gel electrophoresis
The final product UV visualisation
3-4 hours
Cell Debris Removal
by Phenol/Chloroform Extraction
Mix thoroughly with
an equal volume of
organic solvent
e.g. phenol, chloroform,
or phenol:chloroform
Centrifuge Collect aqueous phase
Organic
Aqueous
Interphase
The aqueous phase contains water-soluble
molecules, including nucleic acids.
Proteins and lipids become trapped in the organic
phase, and are thus separated away.
Insoluble debris becomes trapped in the
interphase between the two layers
DNA can be Precipitated with Alcohols
• Pellet down nucleic acids.

• Wash pellet with 70% ethanol to remove
residual salts and other contaminants.

• Discard ethanol and allow pellet to dry.
After
Add alcohol to
precipitate nucleic acids
from the aqueous
fraction
Supernatant
Pellet
70% EtOH
Dissolve pellet
(H
2
O, TE, etc.)
Before
Centrifuge Wash Centrifuge
--> also yields pure DNA, is cheap, but it involves hazardous
chemicals and residual phenol can inhibit PCR and other
enzyme reactions
Polymerase Chain Reaction (PCR)
DNA Sequencing – Dideoxy Method
(Sanger’s Method)
• The dideoxy method gets its
name from the critical role played
by synthetic nucleotides that
lack the -OH at the 3′ carbon
atom.
• A dideoxynucleotide
(dideoxythymidine triphosphate
— ddTTP — is the one shown
here) can be added to the
growing DNA strand but when it
is added, chain elongation
stops because there is no 3′ -
OH for the next nucleotide to be
attached to.
• For this reason, the dideoxy
method is also called the chain
termination method.
Because they lack the -OH (which allows nucleotides
to join a growing DNA strand), replication stops.


Normally, this would
be where another phosphate
Is attached, but with no -OH
group, a bond can not form and
replication stops
The Sanger method requires
• Multiple copies of single stranded template
DNA
• A suitable primer (a small piece of DNA that
can pair with the template DNA to act as a
starting point for replication)
• DNA polymerase (an enzyme that copies
DNA, adding new nucleotides to the 3’ end of
the template
• A ‘pool’ of normal nucleotides
• A small proportion of dideoxynucleotides
labeled in some way ( radioactively or with
fluorescent dyes)
• The template DNA pieces are replicated,
incorporating normal nucleotides, but
occasionally and at random dideoxy (DD)
nucleotides are taken up.
• This stops replication on that piece of DNA
• The result is a mix of DNA lengths, each
ending with a particular labeled
DDnucleotide.
• Because the different lengths ‘travel’ at
different rates during electrophoresis, their
order can be determined.
Steps Of Sequencing
• The DNA to be sequenced is prepared as a single strand. This
template DNA is supplied with a mixture of all four normal (deoxy)
nucleotides in ample quantities and a mixture of all four
dideoxynucleotides, each present in limiting quantities and each
labeled with a "tag" that fluoresces a different color ddATP, ddGTP,
ddCTP, ddTTP and DNA polymerase I.
• Because all four normal nucleotides are present, chain elongation
proceeds normally until, by chance, DNA polymerase inserts a
dideoxy nucleotide instead of the normal deoxynucleotide.
• If the ratio of normal nucleotide to the dideoxy versions is high
enough, some DNA strands will succeed in adding several hundred
nucleotides before insertion of the dideoxy version halts the process.
• At the end of the incubation period, the fragments are separated by
length from longest to shortest. The resolution is so good that a
difference of one nucleotide is enough to separate that strand from
the next shorter and next longer strand.
• Each of the four dideoxynucleotides fluoresces a different color
when illuminated by a laser beam and an automatic scanner
provides a printout of the sequence.
Model of DNA
Sequencing Reaction
Inside the sequencer
Capillary tubes
Sample tray
goes here
Reagents
Automated DNA Sequencing
Terima Kasih