A Facile Method for the Construction of Oligonucleotide Microarrays

Abstract

In recent years, the oligonucleotide-based microarray technique has emerged as a powerful and promising tool for various molecular biological studies. Here, a facile protocol for the construction of an oligonucleotide microarray is demonstrated that involves immobilization of oligonucleotide-trimethoxysilyl conjugates onto virgin glass microslides. The projected immobilization strategy reflects high immobilization efficiency ( approximately 36-40%) and signal-to-noise ratio ( approximately 98), and hybridization efficiency ( approximately 32-35%). Using the proposed protocol, aminoalkyl, mercaptoalkyl, and phosphorylated oligonucleotides were immobilized onto virgin glass microslides. Briefly, modified oligonucleotides were reacted first with 3-glycidyloxypropyltriethoxysilane (GOPTS), and subsequently, the resultant conjugates were directly immobilized onto the virgin glass surface by making use of silanization chemistry. The constructed microarrays were then used for discrimination of base mismatches. On subjecting to different pH and thermal conditions, the microarray showed sufficient stability. Application of this chemistry to manufacture oligonucleotide probe-based microarrays for detection of bacterial meningitis is demonstrated. Single-step reaction for the formation of conjugates with the commercially available reagent (GOPTS), omission of capping step and surface modification, and efficient immobilization of oligonucleotides onto the virgin glass surface are the key features of the proposed strategy.

The Role of Transcriptomics: Physiological Equivalence Based on Gene Expression Profiles

“…The microarray technique is based on the basic principle of DNA Hybridization and uses DNA chips consisting of Please cite this article as Miura et al, Reviews in Agricultural Science, 5:21-35, 2017 https://doi.org/10.7831/ras5.21 ©2017 Reviews in Agricultural Science Probes are deposited onto glass slides via the direct (in situ) synthesis of oligonucleotide probes onto the chip surface using photolithographic methods (Gao et al, 2004) or by deposition methods, which include contact-spotting using pins and deposition by ink jet (Hughes et al, 2001;Sethi et al, 2008). Presently, these arrays comprise mostly 40-to 70-mer oligonucleotides spotted on a glass slide.…”

Herpes Meningoencephalitis: Causes, Diagnosis, and Treatment

“…Microarray is an advance and promising technique for diagnosis of any disease [32]. Tong et al irst time used 16S microarray for detection of neonatal bacterial meningitis using patient's blood [33].…”

Nucleotides and Nucleic Acids; Oligo- and Polynucleotides

“…By this method arrays are reported to have similar stability as those prepared by conventional routes. 12 Oligonucleotides with a poly(T) 10 -poly(C) 10 tail can be immobilised onto unmodified glass surfaces by UV irradiation. 13 Oligonucleotides with terminal phosphate groups will bind strongly to glass surfaces treated with a zirconium phosphonate monolayer, and have been used to detect protein targets, and the use of a polyguanine spacer between the surface and target sequence increases the sensitivity of the array.…”

“…Using the lysine derivative (11) it has been shown that the modified PNA will double strandinvade duplex DNA even at GC-rich sequences. 55 Negative charges have also been introduced into PNA to aid cellular uptake of antisense PNA by addition to the N-terminus of substitutions of either phosphonate glutamine (12) or bis-phosphonate lysine. 56 The backbone analogue (13) based on L-diaminobutyric acid forms a stable structure with (10), which from optical measurements has been assigned a left-handed helix.…”

“…77 PNA is also often used as an antisense agent because it shows enhanced affinity for target nucleic acids, enhanced nuclease resistance, and cellular uptake compared with DNA or RNA. Peptide-PNA conjugates have been used in steric blocking for splice correction in Duchenne muscular dystrophy, 78 and splice correction as a triplex-forming PNA, 79 as an antisense agent conjugated to a cationic lipid to aid cellular uptake, 80 and the negatively charged phosphonate (12) has also been used for antisense. 56 …”