Arrayed primer extension on in situ synthesized 5′ → 3′ oligonucleotides in microchannels

Abstract

Arrays of oligonucleotides synthesized in the 5'-->3' direction have potential benefit in several areas of life sciences research because the free 3'-end can be modified by enzymatic reactions. A Geniom One instrument (febit biomed GmbH, Germany), with integrated chip fabrication, multiplex primer extension, fluorescence imaging, and data analysis, was evaluated for studies of genomic variations. Microchannels used for the array synthesis in Geniom One were not optimized before for the APEX method and, as preliminary experiments demonstrated in this study, the signals were strongly affected by the speed of the process inside reaction channels. Using the two-compartment model (TCM), target binding to feature were quantitatively analyzed, revealing profound mass-transport limitations in the observed kinetics and enabling us to draw a series of physicochemical conclusions of the optimal set-up for the APEX reaction. Some kinetically relevant parameters such as target concentration, reaction time, and temperature were comprehensively analyzed. Finally, we applied the arrays and methods in a proof-of-principle experiment where 36 individuals were typed with 900 oligonucleotide probes (sense and antisense primers for 450 markers), using the ABCR gene as a test system. A new DNA analysis method for studies of genomic variation was developed using this all-in-one platform.

An on-bead tailing/ligation approach for sequencing resin-bound RNA libraries

“…The presence of an enzymatically and chemically accessible 3′-end in immobilized oligonucleotides prepared by standard 3′ → 5′ phosphoramidite synthesis may be attractive for many other applications, from labelling ( 43–46 ) to microarrays ( 47 , 48 ) and genotyping ( 49 ). It should be noted that the overall structure of the RNA-bound beads after tailing bears a strong resemblance to the clonally clustered amplicons that are generated in deep sequencing ( 50 , 51 ): multiple copies of one unknown sequence attached to a bead or to a localized surface area, each with a known primer binding site at its 3′-end.…”

Strategies for Mutation Discovery in Retinitis Pigmentosa: Transition to the Next Generation

“…After exten ding a single base labeled with either of four terminating dideoxynucleotide triphosphates (ddNTPs), the identities of newly incorporated nucleotides are determined. 17,18) Commercially available RP diagnosis kits using APEX technology have been introduced. [19][20][21] There are two representative microarray platforms for genomewide association study (GWAS).…”

A multilevel Lab on chip platform for DNA analysis

“…The possibility to implement a complex genetic protocol, such as Single Nucleotide Polymorphism (SNP) detection, on microdevices has been previously investigated demonstrating the compatibility between materials and reagents Podder et al 2008;Pullat et al 2008;Sundberg et al 2007). Nevertheless a true LOC approach was not implemented.…”