Outline

•Introducing pharmacogenetics in clinical practice

•Basic concepts in genetics and genomics
•Analytical Methods to Identify Genetic Variations
•Genetic variability in drug metabolism: phase I and phase II drug-
metabolizing enzymes
• Drug Transporters
• Pharmacogenomics resources
• Disease-Specific Applications
• Ethical and legal considerations in genetic testing
 Outline
•Introducing pharmacogenetics in clinical practice
•Basic concepts in genetics and genomics
•Analytical Methods to Identify Genetic Variations
•Genetic variability in drug metabolism: phase I and phase II drug-
metabolizing enzymes
• Drug Transporters
• Pharmacogenomics resources
• Disease-Specific Applications
• Ethical and legal considerations in genetic testing
 Sources of DNA Used for Genetic Analyses
• gDNA can essentially be extracted from any part of the
human body.
• The most widely utilized sources of gDNA are:
1. Blood
2. Saliva or,
3. Cheek brushings.
 Sources of DNA Used for Genetic Analyses
• Whole blood: is easy to handle and store and high ­quality
and enough gDNA are typically obtained from specimens as
little as a few drops.
• 200 µl of the whole blood yields 1-12 µg of DNA.
 Sources of DNA Used for Genetic Analyses
• Mouth swabs: the amount of DNA retrieved from such samples may
vary considerably and may allow for only a limited number of genetic
tests to be performed. Also, the integrity of gDNA may be
compromised if swabs or brushes are not immediately processed or
frozen for long ­term storage.
• 1-3 ng/µl in 150 µl human buccal swabs.
 Sources of DNA Used for Genetic Analyses
• Saliva has become more popular as gDNA source as commercial
collection devices have become available that are not only
convenient to use but also stabilize the specimen and allow for long ­
term storage under a variety of conditions making them an excellent
choice for collection outside the clinical or laboratory setting including
patient self ­sampling.
Purification of Genomic DNA, Quantity and
Quality Assessment
• DNA isolation or purification refers to its removal from
other cellular components.
• For many diagnostic analyses or research applications
including DNA sequencing, genotyping, and cytogenetic
testing, to name a few, DNA requires extraction and a
minimum level of purity.
 Purification of Genomic DNA, Quantity and Quality
Assessment
• The following are the essential steps included in a typical
DNA isolation procedure:
1. lysis of the cells containing the DNA (by alkaline lysis, a
detergent, or mechanical disruption),
2. degradation of proteins present in the sample including
those associated with the DNA (using a protease), and
3. separation of the DNA from all other components (via
ethanol precipitation or spin column).
 Lysis and degradation of proteins
• Strong ionic detergents such as sodium dodecyl sulfate (SDS) can
provide cell lysis of the order of seconds, tending to denature proteins
from the cell.
• Proteinase K is also used in the process of nucleic acid extraction to
break down the protein component of the cell membrane and allow
access to the DNA and RNA. It is effective at digesting many types of
proteins, including those that are resistant to other types of
proteases, such as trypsin.
 Lysis and degradation of proteins
• Strong ionic detergents
such as sodium dodecyl
sulfate (SDS) can
provide cell lysis of the
order of seconds,
tending to denature
proteins from the cell.
 Lysis and degradation of proteins
• Proteinase K is also used in
the process of nucleic acid
extraction to break down the
protein component of the
cell membrane and allow
access to the DNA and RNA.
• It is effective at digesting
many types of proteins,
including those that are
resistant to other types of
proteases, such as trypsin.
Separation of the DNA
• Ethanol precipitation or
• Spin column-based
nucleic acid purification is
a solid phase extraction
method to quickly purify
nucleic acids.
Separation of the DNA
• Ethanol precipitation or
• Spin column.
 Purification of Genomic DNA, Quantity and
Quality Assessment
• There are numerous commercially
available kits accommodating wide
ranges of sample types, volumes, and
sizes.
• Different amounts may vary depending
on tissue type, and sample quality.
• Typical amounts of gDNA isolated from
0.2 mL of whole blood, 25 mg of tissue,
and 0.2 mL of saliva are 1–12, 10–50,
and 0.5–10 μg, respectively.
 Purification of Genomic DNA, Quantity and
Quality Assessment
• The overall quality of a gDNA sample is
measured by its molecular integrity and the
presence of impurities such as residual protein.
• The presence of protein can be assessed
spectrophotometrically by the ratio of the
absorbance of UV light at a wavelength of 260
and 280 nm (A260/280). A ratio between 1.7
and 2 generally indicates a high-quality gDNA
sample.
• The drawbacks of spectrophotometry include
the inability to distinguish between DNA, RNA,
and other contaminants, single­and double­
strand DNA, and its relative insensitivity.
 Purification of Genomic DNA, Quantity and
Quality Assessment
• NanoDrop devices can measure
absorbance and/or fluorescence in
small sample volumes of 0.5–2 μL.
 Genotyping
• Is the process of determining
differences in the genotype of
an individual by examining the
individual's DNA sequence
using biological assays and
comparing it to another
individual's sequence or a
reference sequence.
• It reveals the alleles an
individual has inherited from
their parents.
 Alleles
• Two gene copies from
each parent.
• Alleles are various
forms of genes.
• The combination will
predict phenotype and
is called genotype.
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 Genotyping
• Some of the genotyping is
partial (only a small
fraction of an individual’s
genotype is determined).
• New mass-sequencing technologies promise to provide
whole-genome genotyping (or whole-genome sequencing)
in the future.
 Current methods of genotyping
1. Polymerase chain reaction (PCR).
2. Restriction fragment length polymorphisms (RFLPs).
3. Quantitative real-time PCR.
4. DNA sequencing.
5. AmpliChip® CYP450 Test.
6. Genome-Wide Association Studies (GWAS).
7. Detection of Copy Number Variation.
 Methods to detect sequence variations
• Methods or platforms to detect DNA sequence variations
can be divided into:
1. Those designed to identify specific SNPs of interest, for
example, to determine a person's genotype for a given
drug-metabolizing enzyme or
2. A panel of genes, and those supporting genome-wide
analyses.
• All depend on the polymerase chain reaction (PCR).
 Current methods of genotyping
1. Polymerase chain reaction (PCR).
2. Restriction fragment length polymorphisms (RFLPs).
3. Quantitative real-time PCR.
4. DNA sequencing.
5. AmpliChip® CYP450 Test.
6. Genome-Wide Association Studies (GWAS).
7. Detection of Copy Number Variation.
 Polymerase Chain Reaction
• Polymerase chain reaction (PCR): enables researchers to
produce millions of copies of a specific DNA sequence in
approximately two hours.
• The name, polymerase chain reaction, comes from the DNA
polymerase (Taq: Thermus aquaticus) used to amplify
(replicate many times) a piece of DNA by in vitro enzymatic
replication.
• The original molecule or molecules of DNA are replicated by
the DNA polymerase enzyme, thus doubling the number of
DNA molecules.
• To perform a typical PCR reaction,
some sequence information about
two regions of your DNA of interest
is required so that appropriate
primers may be synthesized.
• The two primers, each typically about
15-20 nucleotides in length, are
usually designed so they are 200-
2000 bp apart, one hybridizing to
one strand of dsDNA, the other
hybridizing to the other strand such
that both primers are oriented with
their 3' ends pointing towards each
other.
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 Optimization
• Optimizing the annealing
temperature.
• Optimizing the Mg2+
Concentration.
• Must be confirmed practically.
 Can PCR Amplify RNA?
• Not directly — the DNA
polymerase requires a DNA
template and will not copy
RNA.
• mRNA can first be copied into
cDNA using reverse
transcriptase.
• cDNA is a template for PCR —it
need not be double-stranded.
 Current methods of genotyping
1. Polymerase chain reaction (PCR).
2. Restriction fragment length polymorphisms (RFLPs).
3. Quantitative real-time PCR.
4. DNA sequencing.
5. AmpliChip® CYP450 Test.
6. Genome-Wide Association Studies (GWAS).
7. Detection of Copy Number Variation.
 Restriction Fragment Length Polymorphisms
(RFLPs)
• This method was one of the
earliest employed for
genotyping detecting SNPs.
• It is simple to perform and
requires only basic
equipment.
• However, it is time-consuming due to the enzymatic
digestion and electrophoresis step and cannot be
automated.
 Restriction Fragment Length Polymorphisms
(RFLPs)
• This method exploits a sequence
difference between the wild type and
variant that impacts the recognition site
of a restriction enzyme.
• The region of interest is amplified by
PCR and subsequently incubated with a
restriction enzyme.
• The resulting fragments are then
separated by agarose gel
electrophoresis.
 Restriction Fragment Length
Polymorphisms (RFLPs)
• The length of the restriction fragments is altered if the genetic variant
alters the DNA to create or abolish a restriction site.
• In either case, cleavage with endonuclease results in fragments of
lengths differing from the normal (more or fewer), which can be
detected by DNA hybridization.
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 Current methods of genotyping
1. Polymerase chain reaction (PCR).
2. Restriction fragment length polymorphisms (RFLPs).
3. Quantitative real-time PCR.
4. DNA sequencing.
5. AmpliChip® CYP450 Test.
6. Genome-Wide Association Studies (GWAS).
7. Detection of Copy Number Variation.