1.) DNA Extraction

Follow Kit
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Grind sample
Mix with solution and
spin
Bind, Wash, Elute
Is DNA present?


Gel Electrophoresis
DNA has negative
charge
DNA Replication

In Vivo



DNA unwound
(denatured) by
enzymes
RNA polymerase
makes “primer”
DNA polymerase
adds nucleotides

In Vitro



DNA unwound by
heat
Primers are added
DNA polymerase
adds nucleotides
PCR Protocol

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
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Primer I (reverse)
Primer II ( forward)
DNA template
Taq DNA polymerase
MgCl2
Deoxyribonucleotide triphosphate (dNTP)
PCR buffer
Deionized water
2) Polymerase Chain Reaction



Amplify certain sequence into billionfold
Put DNA sample into tube and mix with
ingredients:
Stages of PCR process



Denature
Anneal
Extension
dNTP (Deoxyribonucleotide
triphosphate)
OH
1) Denaturation
“Unwind Helix”
5’
3’
3’
5’
5’
3’
3’
5’
3’
3’
5’
5’
3’
3’
5’
3’
5’
3’
5’
5’
2) Annealing
Forward Primer
Reverse Primer
3) Extension
Add dNTP’s
PCR Clean-Up
Cycle Sequencing

Same as PCR





Denature
Anneal (forward and reverse primers in separate
reactions)
Extension
dNTP
ddNTP

Different size of DNA fragments
ddNTP

a dideoxyribonucleotide triphosphate
(ddNTP), share the same structure as a
normal dNTP, with the exception of the
3' OH group, which is replaced by an H:
dNTP
OH
“ddNTP”
H
dNTP & ddNTP
…ddNTP



Dideoxynucleotides, or ddNTPs, are nucleotides lacking a 3'hydroxyl (-OH) group on their deoxyribose sugar.
Since deoxyribose already lacks a 2'-OH, dideoxyribose lacks
hydroxyl groups at both its 2' and 3' carbons.
The lack of this hydroxyl group means that, after being added by
a DNA polymerase to a growing nucleotide chain, no further
nucleotides can be added as no phosphodiester bond can be
created based on the fact that deoxyribonucleoside
triphosphates (which are the building blocks of DNA) allow DNA
chain synthesis to occur through a condensation reaction
between the 5' phosphate (following the cleavage of
pyrophospate) of the current nucleotide with the 3' hydroxyl
group of the previous nucleotide.
…ddNTP


The dideoxyribonucleotides do not have a 3'
hydroxyl group, hence no further chain
elongation can occur once this
dideoxynucleotide is on the chain.
This can lead to the determination of the
DNA sequence. Thus, these molecules form
the basis of the dideoxy chain-termination
method of DNA sequencing, which was
developed by Frederick Sanger (1977).
dNTP & ddNTP

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Four separate reaction tubes are required, each one containing
radioactively labeled DNA primers, DNA Polymerase II, and an
ample amount of all 4 dNTP (dATP, dTTP, dGTP, dCTP) each
to be integrated into the DNA strand being synthesized as a
nucleotide.
Each reaction tubes contain a different ddNTP, allowing each
tube to identify a different nucleotide along the strand.
For example, one tube would contain a ddATP, enabling that
reaction tube to identify all the A's being integrated into the
synthesizing strand
Thus all the T's in the complementary template strand (recall
that T nucleotides are complementary and base pair with A
nucleotides).
The above is required for the rest of the tubes with dTTP, dGTP,
dCTP
dNTP & ddNTP

All 4 dNTPs and a different ddNTP are
added to each reaction tube in a ratio of
around 300:1, and Polymerase will
randomly integrate either a dNTP or a
ddNTP into the synthesizing strand if
the ddNTP complements with the
nucleotide on the template strand.
Sequencing method




Before the DNA can be sequenced, it has to be
denatured into single strands using heat.
Next a primer is annealed to one of the template
strands. This primer is specifically constructed so that
its 3' end is located next to the DNA sequence of
interest.
Either this primer or one of the nucleotides should be
radioactively or fluorescently labeled so that the final
product can be detected on a gel (Russell, 2002).
Once the primer is attached to the DNA, the solution
is divided into four tubes labeled "G", "A", "T" and
"C". Then reagents are added to these samples as
….Sequencing method Recap


First, anneal the primer to the DNA template (usually single
stranded), Example:
5'-GAATGTCCTTTCTCTAAG 3'GGAGACTTACAGGAAAGAGATTCAGGATTCAGGAGGCCTA
CCATGAAGATCAAG-5'

Then split the sample into four aliquots, in tubes labeled "G",
"A", "T" and "C" and add the following substrates to the
respective tubes:




"G" tube: all four dNTP's, ddGTP and DNA polymerase
"A" tube: all four dNTP's, ddATP and DNA polymerase
"T" tube: all four dNTP's, ddTTP and DNA polymerase
"C" tube: all four dNTP's, ddCTP and DNA polymerase
…. Sequencing method



When a polymerase (e.g. Klenow fragment) is added
to the tubes, the synthetic reaction proceeds until, by
chance, a dideoxynucleotide is incorporated instead
of a deoxynucleotide.
This is a "chain termination" event, because there is
a 3' H instead of a 3' OH group.
Since the synthesized DNA contains some
radiolabeled (or chemically labeled) substrates, the
products can be detected and distinguished from the
template.
…Sequencing method



As the DNA is synthesized, nucleotides are
added on to the growing chain by the DNA
polymerase.
However, on occasion a dideoxynucleotide is
incorporated into the chain in place of a
normal nucleotide, which results in a chainterminating event.
For example if we looked at only the "G"
tube, we might find a mixture of the following
products:
Figure 1: An example of the potential fragments that could be
produced in the "G" tube. The fragments are all different lengths
due to the random integration of the ddGTP's (Metzenberg).
ddNTP = Array of Fragments

http://www.dnalc.org/ddnalc/resources/c
ycseq.html
Gel Electrophoresis of PCR
Product
Picture of Sequencer
Cycle Sequencing Results

Electropherogram - laser reads DNA
fragment




Adenine
Thymine
Cytosine
Guanine
Electropherogram Example
Electropherogram Figure BAD Results
References






Introduction to Biotechnology by W.J. Thieman and
M.A. Palladino. Pearson & Benjamin Cummings 2nd
edition.
http://en.wikipedia.org
http://www.ocf.berkeley.edu/~edy/genome/sequencin
g.html
http://www.escience.ws/b572/L8/L8.htm
http://www.bio.davidson.edu/Courses/Molbio/MolStud
ents/spring2003/Obenrader/sanger_method_page.ht
m
PPT lecture materials- Courtesy DRs T Mc Elroy &
PN Achar