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ABSTRACTS:
Basis
of Genotyping
Federico
Canzian
Genome Analysis Group, International Agency for Research
on Cancer, Lyon, France
The
next frontier in the genetics of common diseases is to
find genes with high prevalence alleles conferring a low
increase or decrease of risk. This is now possible due
to recent advances of the knowledge of human genetic diversity.
Activities directly or indirectly related to the Human
Genome Project are making available a complete picture
of the variability existing in the human genome, mostly
in the form of Single Nucleotide Polymorphisms (SNPs).
Millions of SNPs of the human genome are already known.
The availability of this knowledge makes it possible to
test a large number of SNPs on a large number of study
subjects. Studying the complete genetic variation in candidate
genes or candidate pathways is within reach by using currently
available technologies. Several technical approaches are
currently used, and a clearly leading one has not emerged
yet.
Many genotyping techniques employ a base extension (a.k.
minisequencing, primer extension etc.) approach. In the
minisequencing reaction, a DNA polymerase is used to specifically
extend a primer that anneals adjacent to the nucleotide
position to be analyzed with a single labeled nucleotide
triphosphate complementary to the nucleotide at the variant
site. Many different ways of detection of the extended
products are then used, including glass microarrays, microspheres,
mass spectrometry and luminometry.
Other approaches rely on hybridization of allele-specific
probes to the stretch of DNA encompassing the variable
nucleotide. These methods are based on hybridization only,
such as high-density microarrays or molecular beacons,
or couple hybridization with enzymatic reactions, such
as the Invader and the Taqman assays.
These different options have specific advantages and disadvantages,
and require a wide range of hardware. The choice of a
genotyping technology depends therefore on considerations
of upfront investments, throughput, running costs, amenability
to automation and availability of data on the validation
of the method. Validation of genotyping data is also a
constant necessity in the course of any genotyping project.
Suitable quality control measures have to be implemented.
A laboratory management information system also has to
be in place to manage, in an automated fashion, the flow
of data generated by a high-throughput genotyping laboratory.
The next development in SNP research is whole-genome association
studies, i.e. to test an appropriate number of markers
over the whole genome in search of novel variants associated
with disease risk. The suitable number of markers to cover
the whole genome depends on the extent of linkage disequilibrium
in the genome and is still under debate, but it is likely
to be in the order of 30,000 to 100,000 SNPs. The main
difficulties to this approach are technical (any study
will have to generate tens of millions of genotypes) and
computational (huge multiple comparisons problem). Possible
approaches to the technical limitations are miniaturization
of genotyping technologies and working on pools of DNAs.
Concerning the computational difficulties, recent work
suggests that large increases in the number of comparisons
have a limited effect on the expansion of required sample
size.
Multiplex
genotyping of SNPs by minisequencing on microarrays: Application
to a pharmacogenetic study
Ulrika
Liljedahl, Katarina Lindroos, Julia Karlsson, Lisa Kurland,
Håkan Melhus, Lars Lind, Ann-Christine Syvänen
Department of Medical Sciences, Uppsala University, 75185
Uppsala, Sweden.
DNA-polymerase-assisted
single nucleotide primer extension, "minisequencing",
allows robust and specific genotyping of SNPs. Consequently
this reaction principle is being applied to SNP-typing
using a variety of assay formats and detection strategies
(reviewed in Syvänen A-C: Hum Mut 13:1, 1999). In
the microarray format of the method multiple primers immobilized
on glass microscope slides are extended with fluorescent
ddNTPs using a DNA polymerase. Recently we have compared
eight chemical methods for covalent immobilization of
the oligonucleotide primers. The best genotyping results
were obtained using mercaptosilane-coated slides attaching
disulfide-modified oligonucleotides (Lindroos K. et al.
Nucleic Acids Res. 29, No 13, e69, 2001.) In our system
the primers are immobilized on standard microscope slides
in a pattern compatible with miniaturised reaction chambers
in a 384-microtiter well format (Pastinen T. et al. Genome
Res. 10:1031, 2000). This assay format allows the generation
of thousands of genotypes per slide. Thus the genotyping
capacity of our system is in practice limited merely by
the ability of multiplexing the PCR (Raitio M. et al.
2001. Genome Res.11:471). We have implemented the system
for genotyping about 100 SNPs in candidate genes regulating
hypertension in DNA-samples from patients from a double-blinded
clinical study. Prior to inclusion of the SNPs in the
detection-panel, we used quantitative minisequencing analysis
of pooled DNA samples (Olsson C. et al. Eur J Hum Genet
8:933, 2000) to rapidly and accurately assess their allele
frequencies in the Swedish population. By solid-phase
minisequencing of the SNPs in individual reactions using
tritium-labelled nucleotides, we are able to detect minor
alleles present at a frequency of 1% in a pooled DNA sample.
Genotyping of this panel of SNPs using TAMRA-labelled
ddNTPs in the multiplex microarray format typically yields
results, with 10 to 100-fold differences in fluorescence
signal ratios between heterozygous and homozygous genotypes.
High-throughput
SNP-analysis by allele-specific primer extension microarrays
Tomi
Pastinen
Montreal Genome Centre, McGill University Health Centre,
Canada
The
increasing demand for high-throughput SNP-scoring calls
for assays that are universally applicable, flexible and
cost-efficient. We have now increased the flexibility
and throughput of our previously described microarray-based
primer extension genotyping (Pastinen T., et al. Genome
Res. 2000;10:1031) by applying generic tag-arrays (Gerry
et al. J. Mol. Biol. 1999;292:251) and multicolour labelling.
Our custom-made tag-arrays are printed in 80 replicates
on a standard microscopic glass slide and each array can
include up to 300 different tags. The genotyping reaction
is performed with 5'complementary tag containing detection
primers and dye-labelled dNTPs or non-fluorescent dNTPs
coupled with "universal" labelling probes. Products
from five extension reactions each labelled with a different
fluorophore are pooled and only legimately extended products
are selectively enriched in purification followed by tag-array
capture and visualization of primer extension products.
Our platform has the potential throughput over 50.000
genotypes per microscopic glass slide and is amenable
to automation.
We are assessing our method in two pilot SNP-association
studies. 30 SNPs in a candidate gene, Delta, known to
harbor variants contributing to bristle number variation
in Drosophila melanogaster are typed in approximately
2000 wild caught flies. The typed SNPs include every common
cSNP at the Delta locus, thus allowing a test of the hypothesis
that the relevant variants contributing to this particular
complex trait are coding versus regulatory in nature.
Second application focuses on SNPs in previously characterized
candidate genes (n=27) for coronary heart disease, which
are assessed in our CHD cohorts derived from the founder
population of North Eastern Quebec. The first-pass results
of the CHD cohort (n=800) demostrated three clusters of
signal-ratios at most of the loci corresponding to the
expected icable multiplex PCR procedures, automation
of signal extraction from the arrays and application of
novel statistical approaches to extract norma SNPs. If the ratio deviates from the expected 50:50 ratio,
the genomic DNA from the sample is then further characterized
to identify putative regulatory SNPs. These functional
SNPs as are potentially more powerful than random SNPs
for large-scale disease association studies. The screening
phase of these "allelic imbalances" in RT PCR
amplified mRNAs requires a quantitative genotyping method.
We are currently evaluating various genotyping approaches
(such as FP-SBE, kinetic ASPCR, kinetic TaqMan and direct
sequencing) in addition to our array platform for their
suitability to high-throughput quantitative allele scoring.
Determination
of SNP Allele Frequencies in Three Defined Populations
and CEPH Pedigrees Using Primer Extension Technology,
SNP-IT, on Multiple Platforms
Michael
Phillips
Orchid Biosciences, Inc., USA
A
key component of the next phase of the Human Genome Project
is the identification, localization and population frequency
determination of single nucleotide polymorphisms (SNPs).
In collaboration with The SNP Consortium (TSC), Orchid
BioSciences is determining SNP allelic frequencies in
samples from three defined populations, and selected CEPH
pedigrees. Forty individuals from each of the Caucasian,
African American and Asian populations are being analyzed
to estimate the frequencies of more than 60,000 TSC SNPs
distributed equally across the genome. The 60,000 TSC
SNPs are also being analyzed on 10 CEPH pedigrees (80
individuals) for the determination of both SNP allelic
frequency and phase. Well over 7 million genotypes will
be determined over the life time of this project using
Orchid's core high throughput single-base primer extension
biochemistry, SNP-IT. The SNP-IT technology will be performed
on multiple platforms. One of the key platforms being
utilized is Orchid's SNPstream 25K platform, which is
based on an automated, single-well assay formatted in
384-well microtiter plates. Another platform that is being
utilized for this project is Orchid's SNPcode, which uses
the Affymetrix universal tag array chip. This platform
can type 500-2000 SNPs per chip and uses a homogenous
primer extension reaction with multiplexed PCR reactions.
Lastly, several other SNP-IT platforms currently being
integrated into the project will be highlighted. Initial
results from the analysis of the TSC SNPs will be presented.
The culmination of these studies will form the basis for
the first defined human SNP map, but more importantly,
these studies will provide the resources necessary to
conduct genome-wide linkage/association studies using
SNPs.
References:
1. Fan JB, Chen X, Halushka MK, Berno A, Huang X, Ryder
T, Lipshutz RJ, Lockhart DJ, Chakravarti A. Parallel genotyping
of human SNPs using generic high-density oligonucleotide
tag arrays. Genome Res. 2000 Jun; 10(6): 853-60
2. Nikiforov, T. T., Rendle, R. B., Goelet, P., Rogers,
Y. H., Kotewicz, M. L., Anderson, S., Trainor, G. L.,
and Knapp, M. R., Genetic Bit Analysis: A Solid Phase
Method for Typing Single Nucleotide Polymorphisms Nuc
Acids Res. 22, 4167-4175 (1994)
3. http://www.orchid.com
4. Riech et al. Linkage disequilibrium in human genome.
Nature. 2001 May; 411; 199-204.
5. Waterstone RH, McPherson JD et al. A map of human genome
sequence variation containing 1.42 million single nucleotide
polymorphisms. Nature. 2001 Feb; 409; 928-933.
Nucleic
Acid Arrays
Mohammad
Sohail
Department of Biochemistry, Oxford University, United
Kingdom
Nucleic acid arrays are a landmark addition to the modern
tools of biological investigation. Although invented only
just over a decade ago, they have become an essential
ingredient of many research proposals. The range of applications
of DNA arrays is wide: some important ones include mutation
detection and the analysis of gene expression. I will
discuss the principles behind some of these applications.
I will also describe the work done in our laboratory on
the selection of antisense reagents and RNA folding using
oligonucleotide scanning arrays.
Genetic
variation, linkage disequilibrium, and the search for
complex disease genes
Lynn
B. Jorde
Eccles Institute of Human Genetics, University of Utah
Health Sciences Center, Salt Lake City, USA
Patterns of human genetic variation have important implications
for the design of studies to locate disease-causing genes.
We present extensive data on worldwide human genetic variation,
using mitochondrial sequences, Y-linked polymorphisms,
and several types of autosomal polymorphisms. These data
all indicate greater genetic diversity in African populations
than in non-Africans, and they indicate that non-African
diversity is a subset of that found in Africa. Our studies
also show that the amount of autosomal variation between
populations within each continent is only 1-2% of the
total variation, implying that population stratification
may not be a serious problem, especially in Asian and
European populations. Our data also support a model in
which modern human populations expanded rapidly in size,
showing that analyses which assume constant population
size are probably inaccurate. We present the results of
resequencing analysis within and near three genes, CYP1A2
(a member of the cytochrome P450 family), IL1B (an interleukin-1
subunit), and AGT (angiotensinogen). Extensive SNP variation
is detected in each of these three genes. A comparison
of our worldwide sample with the NIH DNA Polymorphism
Discovery Resource shows that our sample tends to detect
a larger number of SNPs, but both samples detect the same
common SNPs. As predicted from our studies of worldwide
variation, linkage disequilibrium (LD) is found at greater
distances in Asian and European populations than in African
populations. LD declines with physical distance between
markers, although the pattern of this decline is irregular.
We discuss the implications of these findings for gene-hunting
strategies, and we discuss the relative advantages and
disadvantages of using isolated populations in association-based
gene mapping studies.
SNP
Genotyping by Mass Spectrometry
Ivo
Glynne Gut
Centre National de Génotypage, France
Since
its discovery in 1989 matrix-assisted laser desorption/ionization
mass spectrometry (MALDI) has matured into a powerful
tool for the analysis of biomolecules. Initially it was
applied for the analysis of peptides. Only in the last
five years has it been used for DNA analysis, and become
visible, mainly due to the efforts of Sequenom. Clearly,
the key application in DNA analysis using MALDI is SNP
genotyping. MALDI performs best for the analysis of small
DNA fragments. Analyzing DNA larger than 50 basepairs
is very demanding due to complications of sample purity,
preparation and amounts required.
The CNG has created a high-throughput SNP genotyping platform.
It uses the GOOD assay, a SNP genotyping procedure using
MALDI mass spectrometry. Its noteworthy feature is that,
in contrast to any other published MALDI DNA analysis
methods, it requires no purification. It uses only reagent
additions and incubations for five successive reaction
steps (PCR, shrimp alkaline phosphatase digestion, primer
extension, phosphodiesterase II digestion and alkylation)
in the same tube or microtitre plate. It operates with
the smallest volumes manageable by standard liquid handling
robotics, making it very economical in terms of reagent
consumption. Mass spectrometric analysis yields very high
quality data with full automation for data accumulation
and interpretation. A fully integrated robotic system
with LIMS has been established. Currently it produces
20.000 SNP genotypes per day and is targeted to generate
100.000 SNP genotypes daily in the near future. Data quality
is assessed by a number of control measures. These include
comparison of a subset of samples with sequencing results,
control within CEPH families, Hardy-Weinberg analysis
of each set of 384 SNP genotypes, use of different allele-calling
algorithms and haplotype analysis.
Improvements of the GOOD assay are continually being integrated
for high-throughput. Mainly these are the direct, physical
measurement of the phase of several SNPs (haplotyping)
and the simplification of the GOOD assay to a three-step
procedure, comprising only of PCR, primer extension and
phosphodiesterase II digestion.
DNA-Chip
Technology: Technical Improvements and Application
Jörg
D. Hoheisel
Functional Genome Analysis, Deutsches Krebsforschungszentrum,
Heidelberg, Germany
The
Division of Functional Genome Analysis at the DKFZ is
involved in the development of technologies for the analysis
of large genomic areas to entire genomes with respect
to the encoded functions and their regulation. Based on
technical advances, various functional aspects are being
analysed. One emphasis is work on DNA-, protein- and peptide-microarrays.
Apart from addressing chemical and biophysical issues,
the resulting methods are immediately put to the test
in relevant, biologically driven projects. Also, systems
are being developed toward early diagnosis, prognosis
and evaluation of the success of disease treatment with
some accentuation on cancer. Comparative studies on transcription
and actual protein expression and epigenetic factors are
under way.
(http://www.dkfz-heidelberg.de/funct_genome/index.html)
References:
1. Hauser, N.C., Vingron, M., Scheideler, M., Krems, B.,
Hellmuth, K., Entian, K.-D. & Hoheisel, J.D. (1998).
Transcriptional profiling on all open reading frames of
Saccharomyces cerevisiae. Yeast 14, 1209-1221.
2. Vingron, M. & Hoheisel, J.D. (1999). Computational
aspects of expression data analysis. J. Mol. Med. 77,
3-7.
3. Beier, M. & Hoheisel, J.D. (2000). Production by
quantitative photolithographic synthesis of individually
quality-checked DNA microarrays. Nucleic Acids Res. 28,
e11.
4. Hoheisel, J.D. & Vingron, M. (2000). Transcriptional
profiling: is it worth the money ? Res. Microbiol. 151,
113-119.
5. Frohme, M., Scharm, B., Delius, H., Knecht, R. &
Hoheisel, J.D. (2000). Use of representational difference
analysis and cDNA-arrays for transcriptional profiling
of tumour tissue. Ann. N.Y. Acad. Sci. 910, 85-104.
6. Diehl, F., Grahlmann, S., Beier, M. & Hoheisel,
J.D. (2001). Manufacturing DNA-microarrays of high spot
homogeneity and reduced background signal. Nucleic Acids
Res. 29, e38.
7. Hauser, N.C., Fellenberg, K., Rosario, G., Bastuck,
S., Hoheisel, J.D. & Perez-Ortin, J.E. (2001). Whole
genome analysis of a wine yeast strain. Comp. Funct. Genom.
2, 69-79.
8. Fellenberg, K., Hauser, N.C., Brors, B., Neutzner,
A., Hoheisel, J.D. & Vingron, M. (2001). Microarray
data analysis by correspondence analysis. Proc. Natl.
Acad. Sci. USA, in press.
9. Scheideler, M., Schlaich, N.L., Fellenberg, K., Hauser,
H., Vingron, M., Slusarenko, A.J. & Hoheisel, J.D.
(2001). Monitoring the switch from housekeeping to pathogen
defence metabolism in Arabidopsis thaliana. J. Biol. Chem.
10. Beier, M., Stephan, A. & Hoheisel, J.D. (2001).
Synthesis of photolabile 5'-O-phosphoramidites for the
production of microarrays of inversely oriented oligonucleotides.
Hel. Chim. Acta 84, in press.
11. Matysiak, S., Reuthner, F. & Hoheisel, J.D. (2001).
Automating parallel peptide-synthesis for the production
of PNA-library arrays. BioTechniques, in press.
Fluorescence
detection on DNA-chips
Joachim
W. Engels
Johann Wolfgang Goethe-Universität, Institut für
Organische Chemie
Frankfurt am Main, Germany
Fluorescence detection of oligonucleotides is of prime
importance for DNA-sequencing, DNA-chip analysis and mutation
analysis. A variety of different fluorescent dyes have
been applied based on the laser being used. The appropriate
fluorophores are tethered to the oligonucleotides. This
has mainly been the 5´or 3´-end of the chain.
In addition the base, the phosphate backbone or the sugar
moiety have been utilized as well. Recently we introduced
the possibility to attach the dyes at the exocyclic aminogroups
of A, C and G. These dye conjugated triphosphates are
accepted by different polymerases and seem not to interfere
with the Watson-Crick base pairing. All the labeling strategies
can be incorporated during the standard oligonucleotide
synthesis or done postsynthetically. The advantages and
disadvantages of the different strategies (techniques)
will be discussed.
In addition a novel strategy to circumvent the labeling
of targets during hybridization will be presented.
Fluorescence
Polarization Detection in Nucleic Acid Analysis
Pui-Yan
Kwok
Washington University School of Medicine, St. Louis, Missouri,
63110, USA
When
a fluorescent dye is excited by plane-polarized light,
the fluorescence emitted is also plane-polarized. The
relaxation time associated with the polarized state (that
is, the degree of fluorescence polarization) is determined
by the absolute temperature, the solvent viscosity, and
the molecular volume of the fluorescent dye. When the
temperature and the solvent viscosity are held constant,
the degree of fluorescence polarization is proportional
to the molecular volume of the fluorescent molecule, which
is determined by its molecular weight. Fluorescence polarization
is therefore a good way to monitor significant changes
in molecular weight of fluorescent molecules in a reaction.
Several genotyping assays for single nucleotide polymorphisms
(SNPs) are based on molecular mechanisms that lead to
drastic changes in molecular weight as the reagents are
converted to products. Fluorescence polarization (FP)
is therefore a good detection method for these genotyping
methods. Our group has shown that three SNP genotyping
approaches work well with FP detection. The first approach
is based on the incorporation of a specific dye-terminator
by DNA polymerase. The second approach is based on 5'-nuclease
cleavage of allele-specific probes. The third approach
is basedon invasive cleavage of allele-specific flap probes
by cleavase.
In all three approaches, FP works well and the detection
method reduces the complexity of the starting reagents
without the need for separation or purification at the
end of the assay. FP detection lowers the cost of SNP-specific
probes thereby reduces the cost of assay development.
FP detection is therefore a cost-effective modality in
SNP genotyping.
References:
1. Chen, X, Levine, L, and Kwok, P-Y: Fluorescence polarization
in homogeneous nucleic acid analysis. Genome Res. 1999;9:492-498.
2. Latif, S, Bauer-Sardi?a, I, Ranade, K, Livak, K and
Kwok, P-Y: Fluorescence polarization in homogeneous nucleic
acid analysis II: 5'-nuclease assay. Genome Res. 2001;
11:436-440.
3. Hsu, TM, Law, SM, Duan, S, Neri, BP, Kwok, P-Y: Genotyping
single nucleotide polymorphisms by the Invader assay with
dual-color fluorescence polarization detection. Clin.
Chem. 2001; 47:1373-1377.
4. Hsu, TM, Chen, X, Duan, S, Miller, R, and Kwok, P-Y:
A universal SNP genotyping assay with fluorescence polarization
detection. BioTechniques, in press.
Gene
and genome wide tools for sequence variation management
Heikki
Lehväslaiho
EMBL Outstation, European Bioinformatics Institute, Wellcome
Trust Genome
Campus, Hinxton, UK
I will give an overview of publicly available programs,
web sites and data bases used to analyse and organize
sequence variations. This will be based on work attaching
variations to annotated genomic sequences in and Ensembl
(http://www.ensembl.org/),
and data exchange between HGBASE (http://hgbase.cgr.ki.se/)
and dbSNP (http://www.ncbi.nlm.nih.gov/SNP/)
From
informatics point of view there are two distinct approaches
to sequence variations, genic and genomic, which can be
treated separately but are closely linked. I will describe
efforts to:
1.
Create a framework represent an eukaryotic transcribed
and translated gene in such a way that the effects of
(multiple) sequence changes can easily be traced up and
down DNA, RNA, and AA levels.
2.
Store that information in exhangeabe format and compute
additional descriptive attributes.
3.
Place variations into genomic context using their location
in genomic clones.
The
work is done using BioPerl objects (http://bioperl.org/)
genome annotation system.
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