Introduction

The completion of the Human Genome Project was an important initial step in the exploration of human diversity. However, it is the present world-wide search for individual variation in the human genome that promises to elucidate how genetic variation interacts with the environment to confer individual resistance or susceptibility to disease, responsiveness to medical interventions, and drug toxicity (1). The most common form of genetic variation between individuals is single nucle-otide polymorphisms (SNPs), which are single-base changes at specific DNA sites in the genome, occurring at a frequency of approximately one SNP every 200 to 1000 bp. Different combinations of SNPs in single or multiple genes interact with environmental factors to determine risk for disease as well as variability in how individuals respond to illness and medical therapy and whether they develop adverse drug responses. Research directed toward discovering gene-to-gene and gene-to-environment interaction in disease causation and clinical outcome is increasing at an exponential rate, and pharmacogenomics is often quoted as being poised for application to health care as “personalized medicine” (1,2).

Of the many methods that have been developed for genotyping, those based on the use of microarrays offer the greatest potential for economic, patient-specific application due to their ability to simultaneously interrogate multiple genetic markers (SNPs) using genetic material (template) amplified from an individual using PCR. Genotyping microarrays are devices displaying specific oligonucleotide probes (small lengths of synthetic DNA molecules), precisely located on a small-format solid support such as a glass slide. Although a number of different micro-array genotyping chemistries exist (3,4,5), here we are concerned with arrayed primer extension (APEX) (6). APEX is a minisequencing (7) method based on a two-dimensional array of oligonucle-otides, immobilized via their 5′ ends on a glass surface. The classical APEX probe oligonucleotides (from 15- to 25-mers) are designed so that they are complementary to the gene up to, but not including, the base where the SNP exists, although allele-specific (AS)-APEX oligonucleotide probes (where the 3′ base is the complement of the allelic site) can also be used (8). The S anger-based sequencing chemistry of APEX allows for genotyping of hundreds to a few thousand SNPs, with the array chemistry taking just minutes. APEX achieves this clinically relevant speed because it uses the catalytic ability of a DNA polymerase enzyme to carry out a single nucleotide base extension at the 3′ end of the arrayed oligonucleotide probes, specific to the SNP sites of interest in template DNA that is temporarily hybridized to these probes. Dideoxynucleotide “terminator” bases are prelabeled with fluorophores specific to each one of the four bases of DNA (A, C, G, T). Thus, the fluorescent “color” (wavelength of emitted light) at each of the probe sites (array spots) will give SNP-specific genotypic information.

Scientific literature describing APEX-based and other array-based minisequencing technologies (7) consistently cite the requirement for four fluorescent dideoxynucleotides (with concomitant absence/inactivation of deoxynucleotides) to ensure single-base extension and thus sequence-specific intensity data that can be interpreted as a base call or genotype (3,6). We initially thought that, based on this esTablished dogma of the use of ddNTPs, we could use all four fluorescent dNTPs (instead of ddNTPs) but incorporate them as multiple extensions from each of the oligonucleotide probes because dNTPs would not terminate the DNA polymerization after the initial base extension. For the classical APEX probes, we envisaged that color/base specificity would be lost due to the multiple extensions from the 3′ end of the probe. Thus, these types of probes would no longer be useful for genotyping. However, we predicted that specificity of extension would still be obtained from the allele-specific APEX probes (if an allele was present, the appropriate probe would undergo multiple extensions, rather than just a single extension, and would actually give us greater sensitivity/ higher signal intensity). Overall, this design would allow for a reduction in the costs of genotyping, given the 10-fold higher prices of some ddNTP-conjugated fluorophores compared with dNTP-conjugated fluorophores. We have discovered that whether one uses four fluorescent dNTPs or four fluorescent ddNTPs does not matter to a large degree, and that one still obtains effective single-base extension using either nucleotide reagent type.