APEX arrayed primer extension

APEX is a rapid solid-phase genotyping method that combines the efficiency of a microarray-based assay with Sanger sequencing. In APEX, a DNA microarray (a 'chip') of sequence- (mutation-) specific detection oligonucleotides is used to determine the genotypes in a sample DNA. For each position of interest (e.g. a variable site/SNP) in the sample DNA, two 25-mer oligonucleotides (primers) are synthesized according to the wildtype sequence in both sense and antisense directions. The primers are usually designed with their 3' ends immediately adjacent to the site of interest. The oligonucleotides are arrayed and attached to an amino-activated glass surface via an amino linker at their 5' end with an automated arrayer.

The sample DNA is PCR-amplified in a single or multiplex reaction. All PCR products to be applied to one chip are pooled and purified together. The size of PCR products is not important, because all PCR products will be fragmented before APEX reaction to an optimal size of around 100-200 bp (for subsequent hybridization reaction) by replacing a fraction of dTTP by dUTP in the amplification mix, followed by treatment with thermolabile uracil-N-glycosylase (UNG; Epicentre Technologies, Madison, WI). UNG is highly specific to uracil bases in the DNA; the extent of fragmentation can be, therefore, controlled by the fraction of dUTP incorporation during PCR. The APEX reaction is reliable only if no dNTPs are carried over from the amplification mix, so the dNTP leftover is removed enzymatically by shrimp alkaline phosphatase, in a one-step reaction together with the UNG treatment (3).

Fragmented and heat-denatured PCR product-mix is applied to the chip together with fluorescently labeled ddNTPs (each of the four ddNTPs has a different label) and Thermo Sequenase™ DNA Polymerase (Amersham Biosciences, Piscataway, NJ). During a 15-min hybridization at 58°C, the target sample DNA fragments anneal to the detection primers on the chip immediately adjacent to the queried nucleotide. DNA polymerase extends the 3' end of the primer with a labeled nucleotide analog complementary to the nucleotide of interest resulting in identification of one specific base in the target sequence (Figure 7.1). Covalent bonds between the oligo-nucleotides on the chip and the labeled terminator nucleotides allow a stringent washing of the arrays after hybridization, to minimize the background (4). The signals are acquired by Genorama™ Quattrolmager (Asper Biotech, Ltd.) and Image Pro Plus™ software (Media Cybernetics, Silver Spring, MD) and the genotypes are identified by Genorama™ Basecaller genotyping software (Asper Biotech, Ltd.; Figure 7.2).

An advantage of APEX, compared to purely hybridization-based technologies, is that all nucleotides of interest are identified with optimal discrimination at the same reaction conditions. APEX approach can be successfully applied to the detection of SNPs as well as deletions and insertions in hetero- and homozygous patient samples (Figure 7.2). APEX, performed in a single array format allows for at least one order of magnitude higher discrimination power between genotypes as compared to techniques that are purely hybridization-based (5). APEX technology, as described in this chapter, was developed and is currently provided by Asper Biotech, Ltd, Tartu, Estonia.

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