Lab Manual Section 1: Title Page Volume 2: Molecular assays

Lab Manual Section 1: Title Page Volume 2: Molecular assays
Lab Manual
Section 1: Title Page
Volume 2: Molecular assays
Module name: Single Nucleotide Polymorphism.
Author(s) name/affiliation/email: Carmen Alaez PhD, Clara Gorodezky PhD, DSc. Prof.
Department of Immnunology and Immunogenetics, Instituto de Diagnóstico y Referencia
Epìdemiológicos. MexicoCity .México.
[email protected]; [email protected]; [email protected]
Date prepared or last revised: 09/15/2014
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Section 2 : Comparison of Technical Alternatives
A broad range of techniques are available for SNP genotyping (Slide 11); they differ in
throughput, cost, complexity, equipment, and bioinformatics resources needed etc. Needs are
different in academic laboratories performing research studies versus commercial companies or
genome centers that require high throughput. The type of project to be performed must also be
considered in the selection. For example, genotyping of a small number of SNPs in a large
population could be performed using real-time PCR allelic discrimination assays. However, for
typing of a large number of SNPs on a limited number of individuals, a high throughput system,
such as microarray assays are more adequate.
Choosing a SNP typing platform is not easy, and the following factors should be considered:
(Slide 12)
-Throughput: depending on the study design, the number of SNPs to be typed could vary
from one to a few thousand, and the number of individuals from a hundred to a few
thousands. There are ongoing efforts to increase throughput.
-Simplicity and robustness of the assay: some assays are simple, allow typing of many
different SNPs under similar reaction conditions, and require little optimization. Other
systems require individual reactions to be optimized and run under different conditions.
-Success rate and accuracy: rates of failure or mistyping are variable, and acceptable
levels depend upon the aims of the study and the statistical methods used for data
analysis.
-Track record: Some technologies are new and they do not yet have a well-validated track
record.
-DNA consumption: most methods require only a few nanograms of DNA to type a SNP.
Multiplexing reduces the amount of DNA required, and methods without a PCR step
further reduce further the amount of DNA needed.
- Cost: Some platforms, such as mass spectrometry, require expensive equipment, while
others can be done with standard laboratory equipment. Reagent costs vary greatly
depending on the platform.
We present here the main features of each methodology. Depending on the SNPs available,
commercial kits may be available from various vendors.
Allele specific hybridization (PCR-SSOP): Genotypes can be inferred from the hybridization
signals of allele-specific oligonucleotide probes. The DNA to be tested (most commonly a PCR
product) is fixed to a membrane in a dot or slot format and hybridized with the allele-specific
oligonucleotide probe (referred to as a Dot Blot). Radioactive, enzymatic, and chemiluminescent
systems have been used for detection of the hybridization patterns (Slide 13). A modification of
this format is called reverse dot blot or reverse hybridization, where oligonucleotide probes are
immobilized in a solid phase (membrane, plastic beads, glass etc) and the hybridization is
performed with the DNA to be tested, enabling a higher throughput to be achieved (Slide 14).
High throughput genotyping is the process of identifying the SNP alleles in as many different
individual genomes as possible. This has been achieved using filters or glass slides containing
very high probe densities. Steps of genotyping involve DNA sample preparation, PCR
amplification, and microarray assays. The last improvement in this methodology is the use of DNA
chips with extremely high probe densities that allows one to achieve up to 100,000 genotypes/day
(Slide 15). High throughput genotyping is used to determine haplotypes for SNP mapping. SNP
mapping allows tracking of disease-associated genes and determining the contribution of specific
genes to diseases or phenotypes. Luminex Corporation has developed a panel of up to 200 bead
sets, each of them with unique fluorescent labels that allow bead identification using a flow
2
analyzer. Each bead can be coupled to a DNA probe, to create a bead-based array for
multiplexing genotyping (Slide 16).
Allele discrimination performed on sequence detection systems (SDS) using the TaqMan assay
chemistry also depends on hybridization of two allele-specific probes (Slide 17). The probe for
each allele is labeled with a specific-fluorochrome (FAM or VIC). The oligonucleotide probe also
has a generic nonfluorescent quencher attached to its 3’ end that eliminates background
fluorescence. If both are linked to the oligonucleotide, the fluorescence of the dye is quenched.
During amplification, the 5´ to 3´exonuclease activity of Taq polymerase degrades the probe, but
only if it is hybridized to the template DNA. The fluorophore is released from the quencher, and
the emitted fluorescence can be monitored on a sequence detection system with a real-time PCR
instrument and SDS software. By using two different fluorophores, monitoring can be done in an
allele specific way. Ninety-six or 384-well formats are available and assay design, and
manufacture service is offered by Life Technologies to facilitate the development of assays.
Molecular beacons probes, scorpion probes and dynamic allele-specific hybridization are also
assays based on allele-specific hybridization probes developed by different vendors.
Allele specific digestion (PCR-RFLP-Restriction Fragment length polymorphisms) (Slide
18): Restriction enzymes recognize and cleave specific DNA sequences. A change in any of the
recognition site bases will lead to a failure in the cleavage. Conversely, a mutation in a sequence
closely related to that of a restriction site can create a new site. If the SNP to be tested modifies a
restriction enzyme site, a PCR product containing the SNP could be digested with the
corresponding restriction enzyme. If the PCR product is cut, specific fragments will be generated.
After digestion, they are separated, according to their size by gel electrophoresis, and genotypes
are determined according to the sizes of the digestion fragments obtained. Each SNP allele is
expected to have a different restriction pattern.
Heteroduplex analysis (Slide 19): When alleles in a heterozygote are amplified, and then
denatured and allowed to re-anneal, double-stranded DNA is formed in which one strand is from
one allele, and the complementary strand from the other allele. It thus contains a mismatch at the
SNP site, and has abnormal properties of migration when analyzed by non-denaturing
polyacrylamide gel electrophoresis. Another version of this methodology is denaturing high
performance liquid chromatography (DHPLC) where heteroduplex and homoduplex strands are
separated by their altered retention time on chromatography columns under near denaturing
conditions.
Single Strand conformation polymorphism (Slide 20): This is a popular, simple, and cheap
method of mutation detection, in which changed mobilities of single-stranded mutant molecules
are detected on non-denaturing gels. This technology is based on the fact that only one change
in the DNA sequence is able to produce an altered mobility in electrophoresis, and is not based
on heteroduplex formation.
Primer extension (Slide 21): An oligonucleotide is used to prime DNA synthesis by a DNA
polymerase, as performed in a common PCR reaction. Variations of this method exist. As an
example, for allele specific primer extension, two primers can be used, each of them
complementary in its 3´ end to one of the SNP alleles. DNA synthesis will proceed only if the
3´end of the primer is perfectly matched to template DNA. In this way, an allele-specific PCR
product can be obtained. Allele separation can be achieved using primers labeled with different
dyes, or if the PCR product for each allele has a different size. Amplification refractory mutation
system is another alternative that also involves primer extension.
A modification of this methodology is the single base primer extension (SBE) assay that uses a
single extension base primer. The 3’ end of this primer pair is located exactly before the SNP. The
DNA polymerase is then used to extend one base incorporating the corresponding ddNTPs. Each
of the four ddNTPs is labeled with a different fluorescent dye, allowing detection of the
incorporated nucleotide by gel or capillary electrophoresis on a sequencing apparatus. This
reaction can be multiplexed to type different SNPs under similar conditions at the same time,
reducing cost and increasing the throughput. The different SNPs genotyped simultaneously could
also be separated by hybridization to arrayed tags. Alternatively, the primer to be used can be
3
modified near the 3´end with a charged tag to increase sensitivity to mass spectrometry
detection, and in this case no labeling is necessary.
Oligonucleotide ligation assay (slide 22): Two allele specific oligonucleotides are designed so
they join at the position of the polymorphism to be tested. The oligonucleotides are labeled
differently, and allele discrimination occurs by the ability of DNA ligase to join only perfectly
matched probes; a 3' mismatch in the capture probe will prevent ligation. Detection of the alleles
can be performed by colorimetric methods or by capillary electrophoresis.
Sequence base typing (Slide 23): Direct sequencing of PCR products allows both typing and
identification of new polymorphisms. Pyrosequencing is new sequencing method, based on
primer extension, that produces short segments of sequences (up to 20 nucleotides),
simultaneously on 96 templates. Once the templates have been prepared, 96 of them can be
sequenced in 15 minutes through an automated machine. The method involves sequential
addition of dNTPs to an extension reaction. Incorporation of a nucleotide releases
pyrophosphates that trigger a luciferase-catalyzed enzymatic cascade. The produced light signal
is detected by a CCD camera and is proportional to the number of nucleotides incorporated.
Pyrosequencing is particularly suitable for SNP genotyping. DNA sequencing is the “gold
standard for SNP discovery because it shows all the variation within a sequence. However some
regions are not reliably sequenced because of their high GC content or their secondary structure.
Flap endonuclease discrimination (Slide 24): The “invader” assay involves nuclease cleavage
of a signal probe when two overlapping oligonucleotides hybridize to a complimentary DNA
target. A generic invader probe and an allele-specific primary probe are simultaneously hybridized
to the target sequence such that they overlap at the SNP site. The structure that results is
recognized and cleaved by a flap endonuclease, releasing a probe-specific tail sequence or
“flap”. Flap endonucleases catalyze structure-specific cleavage and are highly sensitive to
mismatches. The cleaved fragment may be labeled with a probe specific fluorescent dye, which
emits light after probe cleavage due to spatial separation from a quencher. PCR amplicons can
be used as the DNA template and the method can also be multiplexed for higher throughput and
cost reduction.
Next Generation Sequencing (NGS) Slide 25: In this methodology, the whole genome or target
regions of the genome, are digested into small fragments. DNA fragments are ligated to platformspecific oligonucleotide adapters that allow laboratories to perform the sequencing biochemistry.
The library of fragments is massively parallel sequences, producing hundreds of gigabases of
data in a single sequence run. The identified strings of bases, called reads, are either aligned to a
reference genome or assembled (de novo sequencing). The full set of aligned reads represents
the entire sequence of the studied region or genome.
Having aligned the fragments of one or more individuals to a reference genome, dedicated
software can identify variable regions and perform an “SNP calling” to determine the genotype for
each the individual.
NGS has a high error rate that can affect accurate SNP and genotype calling. To reduce
uncertainty associated to SNP calling, target regions must be sequenced deeply (20X); however,
deeper sequencing will increase costs. Another alternative is using algorithms that have a
probabilistic framework which incorporates errors that may have been introduced in base calling,
alignment, and assembly, and also uses prior information, such as allele frequencies and patterns
of linkage disequilibrium.
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Section 3 :Specimen Requirements
DNA prepared from the samples to be tested.
Known DNA is to be used as positive controls for each SNP genotype.
Minimum requirements
The DNA to be used in SNP genotyping experiments must:
• Be extracted from the raw material with an optimized protocol
• Not contain PCR inhibitors
• Has an A260/280 ratio greater than 1.7
• Be intact as visualized by gel electrophoresis
• Not have been heated above 60 °C, which can cause degradation
5
Section 4: Protocols
Genotyping SNPs using allele specific hybridization probes labeled using Taq Man
5'-nuclease chemistry and real-time PCR as detection systems
Please refer to the
TaqMan SNP Genotyping Assays Protocol - Applied Biosystems product insert found here:
http://tools.lifetechnologies.com/content/sfs/manuals/cms_042998.pdf
Other SNP genotyping assays are also based on allele-specific hybridization probe principles
that use different strategies for signal generation. For example: molecular beacons probes,
scorpion probes, dynamic allele-specific hybridization assays, FRET probes (Roche
Diagnostics GmbH), etc. These are available from different vendors; details can be found in
these and other web sites: http://www.molecular-beacons.com / www.sigmaaldrich.com,
http://www.genomics.agilent.com.
Principle
For each assay, a unique pair of fluorescent dye detectors is used. One fluorescent dye detector
is a perfect match to the wild type (allele 1) and the other one is a perfect match to the mutation
(allele 2). The change in the fluorescence of the dyes associated with each probes is measured
during the experiments. The actual quantity of target sequence is not determined.
After the automated analysis, the unknown samples are assigned to one of the following groups:
• Homozygotes (samples having only allele 1 or allele 2)
• Heterozygotes (samples having both, allele 1 and allele 2)
 Reagents –
a) Taqman assay(s) for the desired SNP(s).
Note.
TaqMan
assays
for
any
SNP
can
be
ordered
from
http://www.appliedbiosystems.com, and then by choosing the TaqMan® Assays-onDemand SNP Genotyping Products. If the assay for the desired SNP is not ready, you
can use the Assays-by-Design service.
b) TaqMan® genotyping Master Mix. Contains Taq DNA polymerase, dNTPs (with dUTP)
and ROX as passive reference to normalize. Normalization is necessary to correct for
fluorescence fluctuations caused by changes in concentration or in volume. Consult the
vendor manual to choose the master mix according to your assay.
c) Genomic DNA to be tested
d) Appropriate DNA control for each genotype
e) Sterile nuclease-free water
Precautions:
TaqMan Universal PCR Master Mix may cause eye and skin irritation. Exposure may cause
discomfort if swallowed or inhaled. Wear appropriate protective eyewear, clothing, and
gloves.
SNP Genotyping Assay Mix contains formamide. Exposure causes eye, skin, and respiratory
tract irritation. It is a possible developmental and birth defect hazard. Wear appropriate
protective eyewear, clothing, and gloves.

Equipment –
Positive displacement pipette, variable volume: 100-1000L, 0.1-10L, 20-200L (pre
PCR)
Vortex
Centrifuge with adapter for 96-well plates
Microcentrifuge
UV spectrophotometer for DNA quantification
Real time PCR platform
6
Freezer, -20ºC
Refrigerator, 4ºC
 Materials:
-Real-time compatible 96-Well Reaction Plate
-Optical Adhesive Cover
- Pipette tips, with filter plugs
-Splash-free 96 well base
-Microcentrifuge tubes, sterile 1.5-mL
-Power-free gloves
Procedure:
1. Calculate the number of reactions to be performed in each assay.
Note: Include at least two non-template controls and a control DNA for each expected
genotype on each reaction plate for optimal performance.
2. Calculate the volume of each component needed for the total number of reactions
according to the corresponding table below. Consider at least two extra reactions to
compensate for the loss due to reagent transfers.
3. Swirl the bottle of TaqMan Universal Master Mix gently to resuspend.
4. Vortex and centrifuge briefly the reagents in the assay.
5. Prepare the “PCR mix” according to the calculations.
Note: Preparation of the PCR mix should be performed in a pre-PCR area, using dedicated
pipettes, powder free gloves and filter tips.
Allelic discrimination PCR reaction using 40X mix
Reaction Component
Final
concentration
2X TaqMan® Genotyping Master
Mix
40X SNP genotyping Assay Mix
(according to the SNP to be typed)
1X
1X
900 nM each
primer
250 nM for the
probe
Genomic DNA diluted in sterile
nuclease-free dH2O (1-20ng total
amount)
Total
Genomic DNA diluted in sterile
nuclease-free dH2O (1-20ng total
amount)
Total
7
Total volume μL
Example for 100
reactions of 25 μL
1250
0.625
62.5
11.875
NA
25
Allelic discrimination PCR reaction using 80X mix
Reaction Component
Final
concentration
2X TaqMan® Genotyping Master
Mix
80X SNP genotyping Assay Mix
(according to the SNP to be typed)
25 μL
Volume/Well
(one reaction)
12.5
1X
1X
900 nM each
primer
250 nM for the
probe
25 μL
Volume/Well
(one reaction)
12.5
Total volume μL
Ex for 100
reactions
1250
0.3125
31.25
12.1875
NA
25
Note: If a different reaction volume is used, the amounts of each component should be
adjusted accordingly. To prepare 5L reaction for 384 well plates, reduce the amount of each
component proportionally
6. Vortex and centrifuge the tube with the “PCR mix” briefly to spin down the contents and to
eliminate air bubbles.
7. Dispense the appropriate volume of the prepared PCR reaction mix into each well in a
96-well reaction plate (13.1ul for 40X assays and 12.81uL for 80X assays).
8. Add sample DNA (11.88 μL for 40X assays or 12.2 μL for 80X assays) to each well
(typically 10ng total amount/per well is used in our lab). Use a calibrated, positive
displacement pipette to minimize contamination and error. Change tips between samples
to prevent cross-contamination
9. Add the corresponding volume of the control DNA for each genotype, to the designated
wells
10. Add the same amount of sterile nuclease-free dH2O to the non template control wells
11. Cover the reaction plate with an optical adhesive cover.
12. Keep the reaction plate on ice until loading in the real-time PCR system
Thermal Cycler conditions:
Times and
Temperatures
Initial Steps
Pre PCR read Holding Stage
(holding stage)
1 min. to 60° C
10 min. to 95° C
PCR (each for 40 cycles)
Denature
Anneal/extend
15 sec. to 95° C
1 min. to 60° C
Final Step
Post PCR
read
(holding
stage)
1 min. to
60OC
Review the user manual for your real-time PCR instrument for instructions for setting up,
running, and analyzing plates

Interpretation
After the run, the software will analyze, convert, and express the raw data in terms of
fluorescence signal versus wavelength, to pure dye components using the extracted pure dye
standards. After identifying the dye components, the software determines the contribution of
each dye in the raw data and plots the results of the allelic discrimination run on a scatter plot
of Allele X versus Allele Y.
Each well of the 96-well reaction plate is represented with a point on the plot. The clustering
of points can vary along the horizontal axis (Allele X), vertical axis (Allele Y), or diagonal
(Allele X/Allele Y). This variation is due to differences in the extent of reporter dye fluorescent
intensity after PCR amplification. The clustering algorithm used in the analysis software does
not call genotypes when only one cluster is present.
The correlation between fluorescence signals and sequences in the sample is the basis for
results interpretation
A substantial increase in…
VIC® dye fluorescence only
FAM™ dye fluorescence only
Both fluorescence signals
8
Indicates…
Homozygosity for allele 1
Homozygosity for allele 2
Heterozygosity allele 1-allele 2
Limitations for TaqMan SNP Genotyping Assays (Applied Biosystems).
o
o
o
This procedure does not allow identification of a new SNP in a sequence. It is only
intended to perform genotyping of a known SNP.
The actual quantity of target sequence is not determined.
Allelic frequencies between allele SNPs vary between populations. An allele that is
frequent in one population could be completely absent in another population. Thus,
each SNP assay should be validated with a sample coming from the population of
interest.
Troubleshooting and common problems encountered.
See also the corresponding Power Point presentation for examples of trouble allele
discrimination plots
Troubleshooting from the allele discrimination plots:
Problem
POSSIBLE CAUSES
Only one or two cluster are present
Minor allele frequency is too low for sample size
from the tested population
SAMPLE PREPARATION PROBLEMS:
Samples not in equal quantity due to degraded
or incorrectly quantitated DNA, PCR inhibitors
in the sample
ASSAY PROBLEMS: reagents mishandled or
expired. Rox dye not present in PCR master
Trailing clusters
mix. Evaporation. Pipetting errors. Inefficient
mixing and/or insufficient centrifugation
INSTRUMENT PROBLEM: Thermal cycler
poorly calibrated
SOFTWARE PROBLEMS: Rox dye not
designated as the reference dye
GENETIC REASONS: Individual samples have
two null alleles. If an individual sample
consistently clusters with NON TEMPLATE
CONTROL for a particular assay, it may indicate
the presence of a null allele in the sample due
to partial or complete deletion of the gene; SNP
is tri-allelic
SAMPLE PREPARATION PROBLEMS:
Some samples cluster with the NON
Samples not in equal quantity due to degraded
TEMPLATE CONTROLs
or incorrectly quantitated DNA, PCR inhibitors
in sample
ASSAY PROBLEMS: Evaporation. Pipetting
errors. Inefficient mixing and/or insufficient
centrifugation. Insufficient DNA added to the
well, reagent not added to the well
INSTRUMENT PROBLEM: Block contaminated
SAMPLE PREPARATION PROBLEMS:
Samples not in equal quantity due to degraded
or incorrectly quantitated DNA, PCR inhibitors
in sample
ASSAY PROBLEMS: reagents mishandled or
expired. Pipetting errors. Insufficient DNA
All samples cluster with the NON
added to the well, reagent not added to the well.
TEMPLATE CONTROL
INSTRUMENT PROBLEM: annealing
temperatures on the thermal cycler were too
high or too low for the primer or probes due to
poor calibration
AmpliTaq Gold DNA polymerase was not
activated efficiently due to incorrect thermal
9
Cloudy of diffuse clusters
NON TEMPLATE CONTROL
generate high fluorescence signals
that cluster with samples rather than
close to the origin
Sample(s) did not cluster with
specific allele
Samples not in Hardy-Weinberg
equilibrium (expected ratios of each
genotypes not seen)
Some or all data is missing ( no data
shown on the allelic discrimination
plot)
Some or all alleles not called ( X is
shown on the allelic discrimination
plot)
More than three clusters
10
cycler method. Poor calibration of the thermal
cycler.
SAMPLE PREPARATION PROBLEMS:
Samples not in equal quantity due to degraded
or incorrectly quantitated DNA, PCR inhibitors
in sample
ASSAY PROBLEMS: Rox dye not present in
PCR master mix. Evaporation. Pipetting errors
SOFTWARE PROBLEMS: Rox dye not
designated as the reference dye. If only one
cluster is present, allele discrimination plot
incorrectly scaled
ASSAY PROBLEMS: Reagents mishandled or
expired. Contamination due to poor laboratory
practices
SOFTWARE PROBLEMS: Reporter dye
assigned incorrectly
INSTRUMENT PROBLEM: Block contaminated
GENETIC REASONS: Additional SNPs under
the primer. The presence of additional
polymorphisms under the primer led to
inefficient PCR. SNP is tri or tetra allelic. If a
SNP is tri-allelic, six cluster three homozygotes
and three heterozygotes may be seen. In tetra
allelic the pattern is more complicated. Copy
number polymorphisms with different genotypes
in each copy.
SAMPLE PREPARATION PROBLEMS:
Samples not in equal quantity due to degraded
or incorrectly quantitated DNA; PCR inhibitors
in sample
ASSAY PROBLEMS: Contamination.
Evaporation. Pipetting errors. More than one
sample in the well. Inefficient mixing and/or
insufficient centrifugation.
INSTRUMENT PROBLEM: Block contaminated
GENETIC REASONS: Copy number
polymorphism with a different genotype in each
copy. SNP is on X chromosome. If the SNP is
on X chromosome the number of heterozygotes
will be lower than expected. No male will be
heterozygous for the SNP because they have
only one X chromosome
SOFTWARE PROBLEMS: Detectors and
markers set up incorrectly
SOFTWARE PROBLEMS: No marker assigned
to sample. May have checked Omit for the
missing well(s)
SOFTWARE PROBLEMS: NON TEMPLATE
CONTROL task not assigned to NON
TEMPLATE CONTROL wells. Auto call option
not selected. Sample has only two clusters, but
2-cluster calling option not selected. SDS can´t
assign alleles in this case. Sample has only one
cluster, SDS can´t assign alleles in this case.
Outlier sample too far off scale for alleles to be
called for other samples
GENETIC REASONS: Additional SNPs under
the primer. SNP is tri or tetra-allelic. Copy
number polymorphism with a different genotype
Vector cluster (sample data has two
cluster at the same angle)
11
in each copy.
SOFTWARE PROBLEMS: Several assays were
run, but only one marker was assigned.
GENETIC REASONS: Additional SNPs under
the probe
SAMPLE PREPARATION PROBLEMS:
Samples not in equal quantity due to degraded
or incorrectly quantitated DNA; PCR inhibitors
in sample
Section 6.Quality Control/Assurance/ Validation
Purpose:
To define quality control measures used to help ensure the accuracy of SNP
genotyping
Materials:
 Calibration material recommended for your real-time PCR instrument.
 Known DNAs to be used as positive control for each SNP genotype
 Centrifuge with plate adaptor
 Background plate
 Powder free gloves
Procedure:
1. Control positive DNAs for each genotype should be included in every run
2. A minimum of two non template controls should be included in each run; a higher
number of non template controls wells may be used.
3. The concentration of the template DNA should be the same for each well, in order to
have the best genotype plot during the analysis; high variability in DNA concentration
produces dispersion of the plot and difficulty in the interpretation of the results.
4. The accuracy of genotype calls performed should be reviewed by the operator
5. A positive amplification signal in non template control wells is an indication of
reagent contamination or contamination during plate set up; the whole run should be
repeated
6. All reagents should be homogenized before use
7. Fluorophores are light sensitive, thus the assays should be kept on ice and protected
from light.
Recommended Maintenance Schedule for Real-Time PCR instrument
Follow vendor recommendations
12
Section 7.Clinical consideration.
The methodology presented here may be used to type any SNP in the genome. Clinical
consideration and interpretation of the results must be directly related to the purpose of
the study and the specific SNP studied. Variations in DNA sequences can affect disease
development in different autoimmune, infectious, and chronic diseases where a genetic
component has been suggested or an immunological alteration has been documented.
Response to pathogens, chemicals, drugs, vaccines, and other clinical settings may also
have relationships with different SNPs. Nowadays SNP testing is also used in genetic
mapping, pharmacogenetics, pharmacogenomics, genetic testing or functional
proteomics. Recently, knowledge of the SNP content of HLA haplotypes has provided a
means to estimate possible risks prior to hematopoietic stem cell transplantation. In
particular, possible complications may be avoided if donor selection includes matching
SNPs in the MHC region. (Petersdorf EW, et al. Mapping MHC haplotype effects in
unrelated donor hematopoietic cell transplantation. Blood. 2013 PubMed PMID:
23305741).
13
Section 8.Challenges and future Direction.
Over the last 5 years, GWAS (Genome Wide Association Studies) have rapidly and
successfully outperformed candidate gene studies as tools for locating SNP loci as risk
factors in many chronic, autoimmune, and infectious diseases. Susceptibility loci such as
various cytokines and cytokine receptors as well as chemokines, TLRs, TRIMs, and many
others have been identified clearly as genuine classes of autoimmune disease risk
loci..(Baschal EE et al., Congruence as a measurement of extended haplotype structure
across the genome. J Transl Med. 2012 Feb 27; 10:32. doi: (electronic version
publication): 10.1186/1479-5876-10-32; New susceptibility loci associated with kidney
disease in type 1 diabetes. Sandholm N, et al, PLoS Genet. 2012;8: e1002921. Doi
(electronic version publication): 10.1371 A unified approach for allele frequency
estimation, SNP detection and association studies based on pooled sequencing data
using EM algorithms. Chen Q, Sun F. BMC Genomics. 2013 21;14Suppl 1:S1. Doi
(electronic version publication): 10.1186)
Improving the prediction of many diseases in the future will be possible, but the question
remains what the clinical implications of such predictive risk models would be. The
discriminative accuracy that is required in preventive or clinical care depends on the goal
of testing, the availability of (preventive) treatment, and the adverse effects of falsepositive and false-negative test results. Although the early results from GWAS studies
have not yet been used clinically, at least a partial goal of understanding the genetic basis
of these diseases is to investigate the use of these variants to predict disease risk, so that
environmental changes or therapeutic interventions can be initiated before the
inflammatory process progresses or even starts. Also, by better mapping the genetics of
any disease, we hope to improve our understanding of their pathophysiologies. This
understanding may help us find better and new therapeutic drugs. By combining family
history with a quantitative measure of genetic risk, a screening method might eventually
be implemented that could identify clinically silent evidence of disease among first-degree
relatives of patients, who have a high risk of developing the disease. Also improving the
risk prediction would enable us to distinguish individuals at risk to start early treatment for
reducing the accumulation of disability. (Jafari N., Perspectives on the Use of Multiple
Sclerosis Risk Genes for Prediction. Plos ONE, 2011: 12: e26493; Vanderbroeck K et al,
A cytokine gene screen uncovers SOCS1 as genetic risk factor for Multiple Sclerosis.
Genes & Immun., 2012, 13:21)
In conclusion, a broad range of techniques are now available for SNP genotyping,
although they differ in throughput, cost, complexity, equipment and bioinformatics
resources needed. Evolution toward a lower cost, higher simplicity and more powerful
analysis software will be necessary in the future, due to the increasing number of
applications of SNP typing in research, clinics, microbiology and pharmaceutical industry
(High-throughput, high-fidelity HLA genotyping with deep sequencing. Wang C,et al.,
ProcNatlAcadSci U S A. 2012 109:8676;
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