Multiplex, fluorescent, solid-phase minisequencing for efficient screening of DNA sequence variation

Abstract

We developed a multiplex, solid-phase minisequencing method to detect multiple single-nucleotide polymorphisms in an undivided sample. The amplified DNA templates are first captured on a manifold. Then, with multiple minisequencing primers of various sizes, single-nucleotide extension reactions are carried out simultaneously with fluorescently labeled dideoxynucleotides. The size of the extended product, determined by using a DNA sequencing instrument, defines the site of the polymorphisms, and the incorporated nucleotide gives the identity of the nucleotide at each site. HLA-DQA1 typing was used as a model system to evaluate the method. The DR2 subgroup of the HLA-DRB1 gene was typed along with the DQA1 gene to demonstrate the feasibility of the method in analyzing multiple genes at multiple sites simultaneously. The method is generally applicable for screening any single-nucleotide polymorphisms or point mutations, and its manifold format allows practical handling of large numbers of samples.

New Aspects of DNA-based Authentication of Chinese Medicinal Plants by Molecular Biological Techniques

“…New innovative automated assays and specific tools for DNA analysis are emerging and will contribute to the next generation of technologies. These are minisequencing [234,235], nanoscale DNA sequencing [236] or microsphere-based suspension arrays [237]. Further promising developments are the nanopore technology for identification of DNA bases with very high confidence, and the arrayed primer extension reaction (APEX) which is an enzymatic genotyping method to analyze hundreds to thousands of variations in the genome simultaneously in a single multiplexed reaction [238,239].…”

Identification of Species with DNA-Based Technology: Current Progress and Challenges

“…A broad spectrum of highly innovative automated assays have been devised based on conventional genotyping techniques (DNA hybridization or sequencing, real-time PCR, etc) to provide reliable, rapid and low-cost DNA screenings. Methods such as multiplex minisequencing [107,108], microarray primer extension [109], coded microspheres [110] or sequence-coded oligonucleotide ligation assay [111] have a high multiplexing capacity and are rapidly approaching feasible commercial prices [6].…”

The single-nucleotide primer extension (SNuPE) method for the multiplex detection of various DNA sequences: from detection of point mutations to microbial ecology

“…In addition, using different target-specific primers also varying in length allows the analysis of more than one locus (up to 8-12) in one reaction. In the first studies, automated DNA sequencers equipped with four-fluorophore detection systems were used for size separation of the extended products and for the simultaneous detection of the incorporated fluorophore via laser-induced fluorescence [19,20]. Combined with the higher resolution power of capillary electrophoresis as compared with that of gel-based systems, a more accurate size separation of the extended products could be achieved [21][22][23][24].…”