|
 SNP
chips are a type of microarray designed to identify a
set of genetic variants (single nucleotide polymorphisms
or SNPs) in an individual. Currently, we have the ability
to screen over 1000 SNPs for a single individual on a
single microarray slide using the arrayed primer extension
(APEX) method. APEX combines the advantages of a high
throughput parallel microarray-based method with the discriminatory
power of the Sanger dideoxy terminator sequencing. This
combination of hybridization and single base extension
yields greater discrimination and lower background than
hybridization alone.
APEX is a re-sequencing method based on a two-dimensional
microarray of oligonucleotides, immobilized via their
5' ends to a glass surface. For each SNP selected, a minimum
of 6 probes are designed and bound to the slide surface.
The primary (APEX) probes (one per strand) are complementary
up to, but not including, the variant base. Each secondary
(ASO) probe is complementary up to the SNP and ends with
one of the potential variants for that SNP. DNA is PCR
amplified using SNP-specific primers in multiplex reactions.
The products are pooled, purified and fragmented before
hybridizing to the slide-bound oligos. Following this,
a single base extension reaction is performed in-silico.
Slides are scanned and the TIFFs are quantified using
Imagene software and analysed using our in-house software.
A homozygous SNP provides a strong signal from one of
the two APEX probes whereas a heterozygous SNP will have
signal from both probes. Base extension will only occur
with ASO probes complementary to template DNA. Thus, extension
(and therefore probe signal) will occur in 2 ASO probes
for a homozygous SNP and 4 for a heterozygous SNP. This
provides a useful redundancy in the probe set that gives
considerable robustness in the genotyping, and hence confidence
in the data.
Our facility recently developed a genotyping microarray
to screen more than 200 SNPs in control and prostate cancer
patients. Our custom SNP chips design program allows our
customers to select their genes and SNPs of interest and
take advantage of the following services:
• Assistance with SNP selection
• Design of APEX and ASO probes
• Chip printing
• Design of multiplex PCR primers
• Optimization of multiplex PCR reactions
• Performing and optimizing hybridization reactions
• Image acquisition and storage
• Data analysis
|
Customers
provide their own set of oligos and we design, test and,
if required, modify the microarray to achieve customer's
satisfaction.