APEX (arrayed primer extension) technology
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.
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.
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 SoftwareTM 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.