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four nucleotides, A, C, T and G, enabling the
synthesis of roughly 10 15 probes in 4 times 25,
or 100 steps. In this way, it’s possible to make
an array of virtually any size – including arrays
that can examine the entire 3.1 billion bases
in the human genome – in 100 steps or less.
By reducing array feature size, more probes
can be packaged onto the same size surface,
increasing the genetic processing power of
each individual array. For instance, the first
commercial GeneChip products shipped in
1994 had a feature size of 100 microns, that by
2005 have been reduced to 5 microns, allowing
400 times more content on each array.
The high data capacity offered by photolithographic
manufacturing techniques has
provided scientists with tools to study up to
500,000 SNP genotypes per sample analyzed.
For any given SNP of two possible genotypes,
A or B, probes are synthesized on the array
corresponding to both alleles. Following hybridization
of the target to the array, scientists
can then determine whether a SNP is an AA,
AB, or BB genotype by simply analyzing
whether the A allele probes have detected
their complementary sequence, or whether
the B allele probes have detected their complementary
sequence, or whether both have
detected complementary sequences.
500K: Probe set strategy
Every SNP genotype represented on a genotyping
microarray is measured through a
perfect match probe, as well as a mismatch
probe. The mismatch probe serves as an internal
control, accounting for spurious signals
and cross-hybridization. Each probe-pair is
the basic unit used to call a SNP genotype.
However, to ensure highly accurate genotype
calls, GeneChip arrays routinely use multiple
probe pairs to call the genotype for each SNP
represented on the array (Fig. 1).
The first probe pair contains the SNP precisely
in the center of the 25mer sequence. The
remaining probe pairs are positioned, or tiled,
to the right and the left of this central position.
B L I T Z L I C H T
This strategy is used to genotype the A and B
allele of every SNP from both sense and antisense
strands of DNA. The need for high-density
manufacturing technique quickly makes
itself obvious when genotyping large number
of SNPs – more than 10 million probes are used
to genotype the 500,000 SNPs represented on
the Human Mapping 500K Array Set.
500K: Assay
The key to array-based SNP genotyping demanded
an assay that would not require allele
specific amplifications. And much the way a
single assay is used to prepare all transcripts
for whole-genome expression analysis, Affymetrix
developed a whole genome sampling
assay (WGSA) that uses only one primer to
genotype hundreds of thousands of SNPs
distributed throughout the genome 10 . Previous
SNP mapping efforts have been hampered by
the need for locus-specific amplification and
the need for many tens of thousands of PCR
amplifications – an expensive and cumbersome
undertaking.
The WGSA method uses a simple restriction
enzyme to digest genomic DNA, creating various
sizes of DNA fragments, each containing
their respective SNPs. However, only certain
sized fragments are applied to the array, so it’s
critical to design microarray probes against
those SNPs that are present on the DNA fragments.
For example, the Mapping 100K set
uses two separate restriction enzyme reactions,
each of which creates a pool of DNA fragments
containing over 50,000 SNPs to be genotyped.
The same strategy is now being used on the
500K to genotype up to 500,000 SNPs – more
SNPs than ever before possible.
Genotyping up to 10,000 custom SNPs
A new generation of genotyping microarrays
and assays now enable researchers to
perform large-scale genotyping in their own
labs with their own panels of SNPs. Highdensity
genome-wide genotyping of custom
Fig. 1: This microarray probe-set strategy enables scientists to quickly determine whether a genome
contains 2 copies of the A allele (AA), two copies of the B allele (BB) or one copy of each (AB).
SNPs for focused mapping studies or for
candidate-gene association studies requires a
method to simultaneously amplify thousands
of selected SNPs and an equally scalable way
to determine the genotype of each SNP under
study. Newly developed MegAllele assays and
GeneChip microarrays are now being used to
genotype up to 10,000 targeted SNPs using
only a single assay and a single microarray.
These assays offer researchers the ability
to focus on specific regions of the genome
more quickly and in greater detail than ever
before. Researchers studying candidate genes
or other regions of the genome have typically
genotyped relatively few SNPs per experiment
due to cost and ease-of-use hurdles. The new
array-based assay lets scientists successfully
genotype more of the SNPs they want in a
single, flexible assay.
28 | 6. Jahrgang | Nr. 6/2005 LABORWELT
Assay
Molecular Inversion Probe (MIP) technology
enables scientists to amplify up to 10,000 targeted
SNPs in one highly multiplexed experiment
that combines 10,000 different PCR reactions
in a single tube. When the SNP sequence
is amplified during the PCR, a fluorescent base
is incorporated at the variable SNP position,
and depending on the genotype – either an A,
T, C or G – the DNA will contain either a green,
blue, purple or red fluorescent molecule.
Probe strategy
Researchers then use microarrays to detect
each SNP sequence, image the associated
fluorescent color and ultimately
determine the final genotype. Every probe
on the microarray surface is designed to
detect a different SNP by hybridizing to
a DNA sequence that acts as a SNP identifier;
each of the 10,000 PCR primers in the initial
MIP assay contains one of 10,000 unique
DNA sequences that serves as a unique tag
for each of the 10,000 single nucleotide polymorphisms.
The scalable targeted genotyping method
uses a four-color high-resolution scanner to
image the fluorescence associated with each
probe – red corresponds to a thymine genotype,
green to a adenine genotype, blue to a
guanine genotype, and purple to a cytosine
genotype. If a SNP sequence is imaged as a
pure red square, scientists will know that both
alleles of the SNP contain a “T” nucleotide – a
“T/T” genotype – because only the thymine
nucleotides that were incorporated during
the MIP assay had red fluorescence.Likewise,
if the probe lights up as pure green, scientists
know both alleles are an “A” nucleotide – or a
“A/A” genotype – because green fluorescence
corresponds to adenine. If a probe fluoresces
yellow; however, scientists know that the SNP
contains one red “T” allele and one green “A”
allele that combine to make a yellow “T/A”