Surface invasive cleavage assay on a maskless light-directed diamond DNA microarray for genome-wide human SNP mapping

Abstract

The surface invasive cleavage assay, because of its innate accuracy and ability for self-signal amplification, provides a potential route for the mapping of hundreds of thousands of human SNP sites. However, its performance on a high density DNA array has not yet been established, due to the unusual "hairpin" probe design on the microarray and the lack of chemical stability of commercially available substrates. Here we present an applicable method to implement a nanocrystalline diamond thin film as an alternative substrate for fabricating an addressable DNA array using maskless light-directed photochemistry, producing the most chemically stable and biocompatible system for genetic analysis and enzymatic reactions. The surface invasive cleavage reaction, followed by degenerated primer ligation and post-rolling circle amplification is consecutively performed on the addressable diamond DNA array, accurately mapping SNP sites from PCR-amplified human genomic target DNA. Furthermore, a specially-designed DNA array containing dual probes in the same pixel is fabricated by following a reverse light-directed DNA synthesis protocol. This essentially enables us to decipher thousands of SNP alleles in a single-pot reaction by the simple addition of enzyme, target and reaction buffers.

Photolithographic Synthesis of High-Density DNA and RNA Arrays on Flexible, Transparent, and Easily Subdivided Plastic Substrates

“…The profile of hybridization intensities of sequence I and sequence II across the columns is consistent with the expected NPPOC deprotection behavior . A similar approach has been used previously to fabricate DNA sequences of opposite polarity within a single feature . This “2-in-1” strategy is key to placing both primer and template strands within a feature to carry out enzymatic reactions.…”

“…32 A similar approach has been used previously to fabricate DNA sequences of opposite polarity within a single feature. 33 This ‘2-in-1’ strategy is key to placing both primer and template strands within a feature to carry out enzymatic reactions.…”

Genome‐Wide Transcriptional Analysis Reveals the Protection against Hypoxia‐Induced Oxidative Injury in the Intestine of Tibetans via the Inhibition of GRB2/EGFR/PTPN11 Pathways

“…All data can be compared with the resourceful sequences with clear functions [52–54]. We use the data to explore the expressing profiles of intestinal tissues from Tibetans at high altitude.…”

Encrypted Oligonucleotide Arrays for Molecular Authentication

“…Arrays containing two sequences per feature can be produced using a maskless fabrication approach that has previously been applied to single nucleotide polymorphism (SNP) detection and RNA array fabrication. 14,21,22 Briefly, the extent of photodeprotection is controlled at an early step of the fabrication process so that an orthogonal protecting group is installed, typically by coupling a dimethoxytrityl-protected phosphoramidite, on approximately half of the molecules populating a given site. This protecting group remains attached throughout the light-directed synthesis of the first sequence and is then removed so the second sequence can be generated with the remaining fraction of site density.…”