High-density DNA Probe Arrays Research Articles

Photolithographic synthesis of high-density DNA probe arrays: Challenges and opportunities

The continual need for increased manufacturing capacity in the production of GeneChip™ DNA probe arrays, and the expanding use of these arrays into new areas of application such as molecular medicine, has stimulated the development of new chemistries and production methods with higher efficiency and resolution. For current production methods based on contact photolithography, modifications in substrate materials and photoactivated synthesis reagents have provided significant improvements in array performance and information content (≥4×106 sequences∕cm2). An alternative next-generation manufacturing process is also in development, which utilizes photoacid generating polymer films, and automated projection lithography systems. This process has the ability to fabricate arrays with 1 micron feature pitch and smaller, providing an unprecedented sequence density of 108∕cm2 and greater.

European Multicenter Evaluation of High-Density DNA Probe Arrays for Detection of Hepatitis B Virus Resistance Mutations and Identification of Genotypes

Polymorphisms along the hepatitis B virus (HBV) genome have an impact on disease outcome, sensitivity to antiviral treatment, escape from vaccination, and laboratory diagnosis. We have designed a diagnostic tool based on duplex amplification of the whole HBV genome and a high-density DNA chip designed to detect 245 mutations, 20 deletions, and 2 insertions at 151 positions and to determine the genotype of the virus in serum. Assay performances were evaluated with 170 samples, characterized by determination of viral load and sequencing of the Pol, S, and precore genes and the basal core promoter. One hundred fifty-three samples (90%) could be amplified and analyzed by the chip. Only two samples with more than 10(3) genome copies/ml could not be analyzed. Genotype had no impact on analytical sensitivity. Reproducibility studies showed no difference between repeats for codon and genotype determination. Genotype determination by sequencing and the chip were concordant in 148 of 151 samples. Twelve thousand one hundred sixty-one codons were analyzed by both techniques. Only 89.4% could be determined by sequencing, and among the remaining 11,335 codons, 92.8% were identical by sequencing and the chip. Failures to identify an amino acid by the chip were mainly due to reduced hybridization efficiency attributed to unexpected polymorphisms. Optimization of the chip-based reagent for the analysis of the HBV genome is ongoing. This first evaluation showed that DNA chip technology can provide important information in relation to the clinical management of chronic hepatitis B.

High-density DNA probe arrays for identification of staphylococci to the species level

Rapid and accurate identification and speciation of staphylococci clinical isolates is important for predicting medical pathology. We evaluated the ability of a high-density DNA probe array based on 16S rDNA sequences to identify Staphylococcus species. Correct identification was observed for 185 out of the 201 strains (92%). Of the 33 tested species, the array was able to correctly identify 30 of them. The total time required for identification of 4 isolates was 5 h. Such a tool represents a powerful method for routine microbiological diagnostic as well as for epidemiological studies.

Photo-removable protecting groups for in situ DNA microarray synthesis.

High-density DNA probe arrays are finding widespread use in biomedical research and diagnostics because of their ability to simultaneously address large numbers of genes. GeneChip® probe arrays are manufactured by use of photolithography techniques (1)(2)(3)(4). At the base of the current Affymetrix process of synthesis of oligo-DNA probes is the use of nucleoside monomers protected with photo-removable groups (Fig. 1A⇓ ). Irradiation of the partially built oligomer with near-ultraviolet wavelengths (mainly 365 nm) deprotects the terminal hydroxyl group, and the use of appropriate masks allows for control of the sequence of the probe and the size of the features. The sensitivity of DNA to ultraviolet radiation limits the range of wavelengths usable for the photolitographic step to mainly the Hg atomic emission at 365 nm. Therefore, the choice of photo-removable hydroxyl protecting groups depends on the absorbance at this wavelength (4). Our ongoing investigations concerning the fabrication of DNA microarrays have several goals, including increasing the rate and the efficiency of photo-deprotection. The rate of deprotection, or photospeed (PS), is a function of the molar absorptivity of the chromophore at the wavelength of irradiation and the quantum yield of the desired photochemical product formation process. Increasing …

Detection of human immunodeficiency virus type 1 antiretroviral resistance mutations by high-density DNA probe arrays.

Genotypic resistance testing has become an important tool in the clinical management of patients infected with human immunodeficiency virus type 1 (HIV-1). Standard sequencing methodology and hybridization-based technology are the two principal methods used for HIV-1 genotyping. This report describes an evaluation of a new hybridization-based HIV-1 genotypic test of 99 clinical samples from patients infected mostly with HIV-1 subtype B and receiving treatment. This test combines RNA extraction with magnetic silica particles, amplification by nested reverse transcriptase PCR, and detection with high-density probe arrays designed to detect 204 antiretroviral resistance mutations simultaneously in Gag cleavage sites, protease, reverse transcriptase, integrase, and gp41. The nested reverse transcriptase PCR success rates at viral loads exceeding 1,000 copies/ml were 98% for the 2.1-kb amplicon that covers the Gag cleavage sites and the protease and reverse transcriptase genes, 92% for the gp41 amplicon, and 100% for the integrase amplicon. We analyzed 4,465 relevant codons with the HIV-1 DNA chip genotyping assay and the classic sequence-based method. Key resistance mutations in protease and reverse transcriptase were identified correctly 95 and 92% of the time, respectively. This test should be a valuable alternative to the standard sequence-based system for HIV-1 drug resistance monitoring and a useful diagnostic tool for simultaneous multiple genetic analyses.

High-density genechip oligonucleotide probe arrays.

High-density DNA probe arrays provide a highly parallel approach to nucleic acid sequence analysis that is transforming gene-based biomedical research. Photolithographic DNA synthesis has enabled the large-scale production of GeneChip probe arrays containing hundreds of thousands of oligonucleotide sequences on a glass "chip" about 1.5 cm2 in size. The manufacturing process integrates solid-phase photochemical oligonucleotide synthesis with lithographic techniques similar to those used in the microelectronics industry. Due to their very high information content, GeneChip probe arrays are finding widespread use in the hybridization-based detection and analysis of mutations and polymorphisms ("genotyping"), and in a wide range of gene expression studies.

PHOTOLITHOGRAPHIC SYNTHESIS OF HIGH-DENSITY OLIGONUCLEOTIDE PROBE ARRAYS

High-density DNA probe arrays provide a massively parallel approach to nucleic acid sequence analysis that is transforming gene-based biomedical research and diagnostics. Light-directed combinatorial oligonucleotide synthesis has enabled the large-scale production of GeneChip® probe arrays which contain several hundred of thousand oligonucleotide sequences on glass “chips” about one cm2 in size. Due to their very high information content, GeneChip® probe arrays are finding widespread use in the hybridization-based detection and analysis of mutations and polymorphisms (“genotyping”), and in a wide range of gene expression studies. The manufacturing process integrates solid-phase photochemical oligonucleotide synthesis with lithographic techniques adapted from the microelectronics industry. The present-generation methodology employs MeNPOC photo-activatable nucleoside monomers with proximity photolithography, and is currently capable of printing individual 10 μm2 probe features at a density of 106 probes/cm2.

HIGH CAPACITY SUBSTRATES AS A PLATFORM FOR A DNA PROBE ARRAY GENOTYPING ASSAY

Colloidal silica particles were deposited on a glass substrate to produce high-capacity porous supports for high-density DNA probe arrays. Porous surfaces were used to increase the addressable surface area and number of probes available for hybridization. Surfaces derived from 70–100 nm size particles deposited in films from 0.15 to 2 microns thick exhibited excellent performance in light-directed oligonucleotide synthesis. Evaluation of these substrates in a genotyping assay is reported.