Multiple primer extension by DNA polymerase on a novel plastic DNA array coated with a biocompatible polymer

Abstract

DNA microarrays are routinely used to monitor gene expression profiling and single nucleotide polymorphisms (SNPs). However, for practically useful high performance, the detection sensitivity is still not adequate, leaving low expression genes undetected. To resolve this issue, we have developed a new plastic S-BIO® PrimeSurface® with a biocompatible polymer; its surface chemistry offers an extraordinarily stable thermal property for a lack of pre-activated glass slide surface. The oligonucleotides immobilized on this substrate are robust in boiling water and show no significant loss of hybridization activity during dissociation treatment. This allowed us to hybridize the templates, extend the 3′ end of the immobilized DNA primers on the S-Bio® by DNA polymerase using deoxynucleotidyl triphosphates (dNTP) as extender units, release the templates by denaturalization and use the same templates for a second round of reactions similar to that of the PCR method. By repeating this cycle, the picomolar concentration range of the template oligonucleotide can be detected as stable signals via the incorporation of labeled dUTP into primers. This method of Multiple Primer EXtension (MPEX) could be further extended as an alternative route for producing DNA microarrays for SNP analyses via simple template preparation such as reverse transcript cDNA or restriction enzyme treatment of genome DNA.

Cell membrane-inspired phospholipid polymers for developing medical devices with excellent biointerfaces

“…For example, a PMBN surface is useful for DNA immobilization, and is applied to produce DNA chips such as Prime Surface R by Sumitomo Bakelite Co. Ltd. Kinoshita et al focused on the hybridization properties with regard to a suitable surface chemistry for a cyclic olefin copolymer (COC) surface with PMBN. They also discussed new approaches for the application of an on-chip DNA detection method through multiple primer extension (MPEX) by DNA polymerase [67]. Besides the nitrophenyloxycarbonyl group, other functional groups were also proposed for the conjugation of proteins with MPC polymers [20,21,68].…”

Plastic Polymers for Efficient DNA Microarray Hybridization: Application to Microbiological Diagnostics

“…Polymer-based slides of PMMA-VSUVT and Zeonor 1060R were fabricated via injection molding (2,10,19,25). Carboxylic acid groups were generated at the surfaces of PMMA-VSUVT slides by immersing the slides in a saturated aqueous solution of sodium hydroxide for at least 10 h. This surface treatment effectively induces hydrolysis of the methyl ester groups at the surfaces of the PMMA slides.…”

Recent developments in nucleic acid identification using solid-phase enzymatic assays

“…An original approach describing a solid-phase amplification with only one surface immobilized primer, and in the absence of a liquid-phase primer, has been described by Kinoshita et al [44]. In this work they tethered a single primer to a solid surface covered with the biocompatible phospholipid polymer, poly [2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-nitrophenyloxycarbonyl polyethyleneglycol methacrylate (MEONP)] (PMBN).…”

“…As a model target a 50 nucleotide long synthetic DNA was used. The workflow consisted of 30 repetitive cycles of hybridization of the DNA target to the immobilized primers which were then elongated with Taq DNA polymerase followed by thermal denaturation [44]. The detection of this primer extension was monitored by the incorporation of Cy-3 labelled dUTP's.…”