Biomedical Engineering Reference
In-Depth Information
3.2 Methods and approaches
3.2.1 Arrays
Different methodologies of SNP typing and types of commercially available SNP arrays have
been developed. Basically, two types of array exist: locus-specific arrays of oligonucleotides
and arrays with universal capture oligonucleotides. The SNP typing assays include method-
ologies such as allele-specific primer extension and whole genome sampling. We will briefly
discuss four different methods. Two different genotyping methods, molecular-inversion
probe (MIP) genotyping and Goldengate genotyping, are based on high level multiplex poly-
merase chain reaction (PCR) with universal primers in combination with universal arrays.
Goldengate genotyping makes use of a multiplex mixture of probes for 96, 384, 768 or
1536 SNPs per array [10]. For each SNP, a combination of allele-specific and locus-specific
primers is annealed to the SNP locus. These primers are tailed with common forward
and reverse primers, and a universal capture probe complementary to the locus-specific
primer. The small gap between the allele and locus-specific probes is filled by subsequent
allele-specific primer extension and sealed by a ligase, resulting in an allele-specific artificial
PCR template. This template is then PCR amplified using fluorescently labeled universal
PCR primers. The resulting probe is hybridized to an array of universal-capture probes and
the array is scanned in a special reader, generating two fluorescent signals representing the
two different alleles of an SNP. MIP genotyping utilizes a pool of locus-specific probes
with a multiplexing degree of over 10 000 SNPs per array. The 5
and 3
ends of each
circularizable probe anneal upstream and downstream of the SNP. The 1 bp gap is filled
in a different reaction for each nucleotide. The probes are subsequently circularized using
ligase to seal the remaining nick; non-annealed and non-circular probes are removed by
exonuclease treatment. Restriction and digestion then releases the circularized probe and
the resulting template is PCR amplified using common primers [11]. The four nucleotide
reactions are labeled in different colors and pooled. Subsequently, the pool is hybridized to
an array of universal-capture probes and the four colors are read out. Genechips can detect
up to a million SNPs on a single chip. For each SNP, a set of locus-specific 25-mer oligonu-
cleotides is present on the array. The sample is prepared according to the whole-genome
sampling assay [12], a method in which the genomic complexity is reduced through restric-
tion enzyme (RE) treatment of high-quality genomic DNA and ligation of a common adaptor
to the digested DNA. The subsequent single primer PCR step results in the reduction of
the genomic complexity through efficient size-selection in the PCR reaction. The prod-
uct is hybridized to a locus-specific array. The SNPs on the array are selected from the
DNA that is represented after the complexity reduction PCR [13]. Finally, Infinium arrays
are locus-specific arrays with allele-specific capture probes. In the assay, genomic DNA is
whole-genome amplified and subsequently fragmented. The resulting probes are then dena-
tured and hybridized to the array. An 'on the array'-allele-specific primer extension assay is
followed by staining and read out using standard immunohistochemical detection methods.
Currently, these arrays can detect over a million SNPs on a single array [14].
3.2.2 Genotyping
After scanning of the SNP arrays, the signal intensities have to be converted into genotype
calls. SNP calling software is available for each platform: Beadstudio for Goldengate and
