Issue 12, 2009
From the journal:

Analyst

A gel-based solid-phase amplification and its application for SNP typing and sequencing on-chip

Huan Huang,†ab   Pengfeng Xiao,†d   Zongtai Qi,ab   Ying Bu,a   Wenbo Liua  and  Guohua Zhou*ac  

* Corresponding authors

a Huadong Research Institute for Medicine and Biotechnics, Nanjing, China
E-mail: ghzhou@nju.edu.cn
Fax: +86 25 84514223
Tel: +86 25 84514223

b China Pharmaceutical University, Nanjing, China

c Medical School, Nanjing University, Nanjing, China

d State Key Laboratory of Bioelectronics, Southeast University, Nanjing, China

Abstract

As conventional solid-phase amplification (SPA) on a two-dimensional slide has a low amplification capacity due to a limited amount of immobilized primers, we propose a three-dimensional SPA by immobilizing primers in hydrogel attached to a slide. One of the PCR primers, modified with an acrylamide group at the 5′-terminal, was copolymerized with both polyacrylamide gel and an acryl-modified glass slide, resulting in a high amplification capacity. The immobilization process was carried out by adding the catalysis reagent N,N,N′,N′-tetramethylethylenediamine (TEMED) volatilized in vacuum, with uniform sample-concentration and gel-viscosity in the course of one-step nucleic acid immobilization. The porous structure of polyacrylamide gel, which allows PCR reagents such as Taq DNA polymerase, primers, dNTPs and DNA templates to freely enter the gel matrix, provides a homogeneous solution-mimicking environment for SPA on the interface or the inside of gel pads. Based on gel-based SPA, genotypes of different samples were accurately discriminated by either dual-color fluorescence hybridization or BAMPER (Bioluminometric Assay coupled with Modified Primer Extension Reactions). Pyrosequencing was also successfully carried out on SPA products. As the linkage between DNA molecules and gel is very strong, SPA products immobilized on gel pads could be reused several times if extended strands were removed by electrophoresis. Thus, the gel-based SPA provides a powerful tool for directly using on-chip amplicons for parallel detection.

Graphical abstract: A gel-based solid-phase amplification and its application for SNP typing and sequencing on-chip

About
Cited by
Related
Download options Please wait...

Article information

DOI
https://doi.org/10.1039/B915121D
Article type
Paper
Submitted
28 Jul 2009
Accepted
25 Sep 2009
First published
05 Oct 2009

Analyst, 2009,134, 2434-2440

Permissions

Request permissions

A gel-based solid-phase amplification and its application for SNP typing and sequencing on-chip

H. Huang, P. Xiao, Z. Qi, Y. Bu, W. Liu and G. Zhou, Analyst, 2009, 134, 2434 DOI: 10.1039/B915121D

To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements