|
PCR and DNA FRAGMENTATION
Polymerase
chain reaction (PCR) is used to amplify regions of genomic DNA which
harbor mutations or polymorphisms (SNPs) of interest. PCR primers are
selected with the help of Chip Design Software.
A fraction of the dTTP-s is replaced by dUTPs in the PCR mix
allowing for later fragmentation with Uracil N-Glycosylase (UNG). The
products are pooled, concentrated and purified enzymatically from
unincorporated dNTP-s using shrimp Alkaline Phosphatase (sAP). After
sAP and UNG treatment the samples are heated to deactivate the enzymes
and to cleave the DNA at uracil sites.
Fragmentation
of the long PCR products facilitates proper hybridization to the
complementary oligonucleotides, which have been immobilized on the
glass.
APEX REACTION
Immediately before APEX reaction, the fragmented PCR products are denatured and
transferred to the previously designed chip (oligonucleotide array on
glass) in a reaction mixture. The buffered reaction mixture contains
single-stranded DNA, thermostable DNA polymerase and four different
terminator nucleotides each tagged with an individual fluorophore.
The
template-dependent DNA polymerase reaction is carried out at elevated
temperature in order to minimize the formation of undesirable secondary
structure in the oligos, yet still to permit efficient hybridization
with the target DNA and not to compromise polymerase activity. After
incubation, the unattached and non-covalently bound material is washed
away, leading to an excellent signal to noise ratio.
In
the oligonucleotide array each spot comprises of a high copy number of
the same immobilized synthetic oligonucleotide designed to determine
one specific nucleotide on genomic DNA. It is important that the
oligonucleotides are covalently bound to the coated slides surface via
an amino linker their 5' ends, leaving their 3' ends of primers free
for the single base primer extension reaction.
DETECTION
The processed slides harbouring an APEX array are imaged in our microarray detector - Genorama® QuattroImagerTM.
Four
lasers (one at a time) are used to excite the different dyes. The four
spectrally well separated dyes are excited via total internal
reflection of the laser beam in the glass slide, which acts as a light
guide. The light emitted by flourophores in response to the excitation is captured using a charge-coupled device (CCD) camera
Since
four different dyes per reaction are used, four different images of
emitted light per array will be recorded. Each image corresponds to one
individual dye, hence reflects to the pattern of the incorporation of
one of the four terminator nucleotides on the array (see inserted
images A, C, T, G on the diagram).
IMAGE and DATA ANALYSIS
Imaging is followed by analysis with Genorama® Genotyping Soft-wareTM to convert the fluorescence information into sequence data. First, the
signal intensities of respective dye terminator images are normalized.
The intensities of four images at all the oligonucleotide positions are
compared and the strongest signal is base called. Image analysis
includes quality control steps. |