This document discusses pharmacogenetics and opportunities for pharmacists in this emerging field. It describes how molecular genetics can help understand disease pathogenesis and monitor drug therapy responses. Pharmacogenetics utilizes techniques like PCR, DNA fingerprinting, and gene therapy to diagnose diseases and develop personalized treatment approaches. The polymerase chain reaction is discussed as a method to amplify DNA sequences, enabling analysis of minute DNA samples. Overall, the document argues that pharmacists should learn about these molecular genetics technologies to better apply them in therapeutic contexts.
Pharmacogenetics: The Emerging Role of Pharmacists in Personalized Medicine
1. 1
Dr. V. S. PAWAR
M.Pharm (Pharmacology) Ph.D
Dept of Pharmacology
SVPM’s College of Pharmacy
Malegaon (Bk), Baramati 413115.
Email: vinod_pharmacology@yahoo.co.in
vinodspawar@gmail.com
Pharmacogenetics: Emerging field for pharmacy
3. 3
Pharmacy profession - ability to adopt technology
not as sideline, but as an integral part
Molecular genetics offer the ability to understand
not only the molecular basis of disease, but the
ability to monitor the response to drug therapy.
It is important to discuss the impact of this
technology and encourage involvement of
pharmacists in the application of molecular
genetics to therapeutics.
Opportunities to Pharmacist
7. 7
The Polymerase Chain Reaction
(PCR) was not a discovery, but
rather an invention
A special DNA polymerase
(Taq) is used to make many
copies of a short length of DNA
(100-10,000 bp) defined by
primers
Kary Mullis, the inventor of
PCR, was awarded the 1993
Nobel Prize in Chemistry
9. What PCR Can Do ?
Starting with one original copy an almost infinite number of
copies can be made using PCR
“Amplified” fragments of DNA can be sequenced, cloned,
probed or sized using electrophoresis
Defective genes can be amplified to diagnose any number of
illnesses
Genes from pathogens can be amplified to identify them (i.e.,
HIV, Vibrio sp., Salmonella sp. etc.)
Amplified fragments can act as genetic fingerprints
10. Polymerase Chain Reaction (PCR)
PCR is a technique which is used to amplify the number of copies
of a specific region of DNA, in order to produce enough DNA
to be adequately tested.
The purpose of a PCR is to make a huge number of copies of a
gene.
As a result, it now becomes possible to analyze and characterize
DNA fragments found in minute quantities in places like a drop
of blood at a crime scene or a cell from an extinct dinosaur.
13. 13
-50 mM KCl,
-10 mM Tris-HCl (pH 8.4),
1.5 mM MgCl2 ,
100 g/ml gelatin
PCR BUFFER
14. 14
PRIMERS
- Primers are the most important components
that determine the success or failure of an
amplification reaction.
- Generally 20-30 bases in length and have
sequence complementary to the target region
- Extension at 3’end.
15. 15
DEOXYRIBONUCLEOTIDES
- dNTPs bind Mg2+ ..
- Usually used at concentrations of 20-200 M.
- High concentrations may promote mis
incorporations
- Lowest dNTP concentration appropriate for
the length and composition of target is chosen
16. 16
Taq POLYMERASE
- Isolated from Thermus aquaticus
- If enzyme concentration is too high, non
specific background products may accumulate
- If too low, insufficient amount of product is
made
21. An agarose gel is prepared
by combining agarose
powder and a buffer
solution.
Agarose
Buffer
Flask for boiling
•Sweetened agarose gels have
been eaten in the Far East since
the 17th century.
•Agarose was first used in biology
when Robert Koch* used it as a
culture medium for Tuberculosis
bacteria in 1882
*Lina Hesse, technician and illustrator for
a colleague of Koch was the first to
suggest agar for use in culturing bacteria
23. Agarose Buffer Solution
Combine the agarose powder and buffer solution. Use a flask that is
several times larger than the volume of buffer.
24. Agarose is insoluble at room temperature (left).
The agarose solution is boiled until clear (right).
Gently swirl the solution periodically when heating to allow all the grains of agarose
to dissolve.
***Be careful when boiling - the agarose solution may become superheated and
may boil violently if it has been heated too long in a microwave oven.
Melting the Agarose
25. Allow the agarose solution to cool slightly (~60ºC) and then carefully
pour the melted agarose solution into the casting tray. Avoid air
bubbles.
Pouring the gel
26. When cooled, the agarose polymerizes, forming a flexible gel. It should
appear lighter in color when completely cooled (30-45 minutes).
Carefully remove the combs and tape.
28. buffer
Add enough electrophoresis buffer to cover the gel to a depth of
at least 1 mm. Make sure each well is filled with buffer.
Cathode
(negative)
Anode
(positive)
wells
DNA
29. Loading the Gel
Carefully place the pipette tip over a well and gently expel the sample.
The sample should sink into the well. Be careful not to puncture the
gel with the pipette tip.
30. Place the cover on the electrophoresis chamber, connecting the electrical
leads. Connect the electrical leads to the power supply. Be sure the leads
are attached correctly - DNA migrates toward the anode (red). When the
power is turned on, bubbles should form on the electrodes in the
electrophoresis chamber.
Running the Gel
31. wells
Bromophenol Blue
Cathode
(-)
Anode
(+)
Gel
After the current is applied, make sure the Gel is running in the correct
direction. Bromophenol blue will run in the same direction as the DNA.
DNA
(-)
32. • DNA is negatively charged.
+-
Power
DNA
• When placed in an electrical field, DNA will migrate toward the positive
pole (anode).
H
O2
• An agarose gel is used to slow the movement of DNA and separate by size.
Scanning Electron Micrograph of
Agarose Gel (1×1 µm)
• Polymerized agarose is porous,
allowing for the movement of DNA
33. +-
Power
DNA
How fast will the DNA migrate?
strength of the electrical field, buffer, density of agarose gel…
Size of the DNA!
*Small DNA move faster than large DNA
…gel electrophoresis separates DNA according to size
small
large
Within an agarose gel, linear DNA migrate inversely
proportional to the log10 of their molecular weight.
37. 37
Staining the Gel
***CAUTION! Ethidium bromide is a powerful mutagen and is
moderately toxic. Gloves should be worn at all times.
• Ethidium bromide binds to DNA and fluoresces under UV light,
allowing the visualization of DNA on a Gel.
• Ethidium bromide can be added to the gel and/or running buffer
before the gel is run or the gel can be stained after it has run.
39. 1. SOUTHERN BLOT
2. NORTHERN BLOT
3. WESTERN BLOT
40. Blotting: History
Southern Blotting is named after its inventor, the
British biologist Edwin Southern (1975)
Earned Sir Southern a Lasker Award in 2005
41. History/Background
Spawned naming of related techniques:
Southern blot
(DNA)
Northern blot
(RNA)
Western blot
(Protein)
Eastern blot
(???)
42. Weight
Glass Plate
Whatman 3MM paper
Gel
Paper towels
Membrane (nylon
or nitrocellulose)
Whatman 3MM
paper
Transfer buffer
43. Buffer drawn from
a reservoir passes
through the gel
into a stack of
paper towels
DNA eluted from
the gel by the
moving stream of
buffer is
deposited onto a
membrane
weight tight connection
45. Matching of DNA
obtained from
Crime scene
Suspects
Victim
SuspectA
SuspectB
SpermDNA
From crime scene
Note:
•Multiple minisatellites will be used in
actual criminal investigation to
increase accuracy
•Crime scene DNA is often degraded and
it may affects the accuracy of RFLP-
based DNA fingerprinting
•Restriction fragment too large
•Sensitive to DNA degradation
•Solved by PCR-based DNA
fingerprinting
46. V S S1 S2 S3
V Victim
S Sample from crime scene
S1 Suspect 1
S2 Suspect 2
S3 Suspect 3
More than 20 fragments
from Suspect 1 match those
taken from the crime scene
DNA Profiles
47. Starting position of sample
1 2 3 4
1 mother
2 child
3 possible father A
4 possible father B
There is a match between one of
the child’s restriction fragments
and one of the mother’s.
There is also a match between the
child’s other fragment and one from
possible father A.
Neither of the child’s restriction
fragments match those of possible
father B
Paternity test
Paternity dispute
48. 48
Dr. Lalji Singh
Vice-Chancellor,
BHU.
(Awarded Padmashri by the President)
Developed a probe called Bkm-derived probe for
DNA fingerprinting which brought CCMB to
limelight. Since then this probe is being
extensively used for forensic investigation,
paternitiy determination and seed stock
verification
49. Gene therapy
Imagine that you
accidentally broke one of
your neighbor's windows.
• Stay silent: no one will ever find
out that you are guilty, but the
window doesn't get fixed.
• Repair it with some tape: not the
best long-term solution.
• Put in a new window: not only do
you solve the problem, but also
you do the honorable thing.
Disease ! Due to gene flaws or
mutations…. Our broken window
• Stay silent: ignore the genetic
disorder and nothing gets fixed.
• Try to treat the disorder with drugs
or other approaches: depending
on the disorder, treatment may or
may not be a good long-term
solution.
• Put in a normal, functioning copy
of the gene: if you can do this, it
may solve the problem!
52. What is Gene Therapy
It is a technique for correcting defective genes
that are responsible for disease development
There are four approaches:
1. A normal gene inserted to compensate for a
nonfunctional gene.
2. An abnormal gene traded for a normal gene
3. An abnormal gene repaired through selective
reverse mutation
4. Change the regulation of gene pairs