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- Pastinen, T, et al.
(författare)
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Minisequencing : a specific tool for DNA analysis and diagnostics on oligonucleotide arrays
- 1997
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Ingår i: Genome Research. - : Cold Spring Harbor Laboratory. - 1088-9051 .- 1549-5469. ; 7:6, s. 606-614
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Tidskriftsartikel (refereegranskat)abstract
- We describe a method for multiplex detection of mutations in which the solid-phase minisequencing principle is applied to an oligonucleotide array format. The mutations are detected by extending immobilized primers that anneal to their template sequences immediately adjacent to the mutant nucleotide positions with single labeled dideoxynucleoside triphosphates using a DNA polymerase. The arrays were prepared by coupling one primer per mutation to be detected on a small glass area. Genomic fragments spanning nine disease mutations, which were selected as targets for the assay, were amplified in multiplex PCR reactions and used as templates for the minisequencing reactions on the primer array. The genotypes of homozygous and heterozygous genomic DNA samples were unequivocally defined at each analyzed nucleotide position by the highly specific primer extension reaction. In a comparison to hybridization with immobilized allele-specific probes in the same assay format, the power of discrimination between homozygous and heterozygous genotypes was one order of magnitude higher using the minisequencing method. Therefore, single-nucleotide primer extension is a promising principle for future high-throughput mutation detection and genotyping using high density DNA-chip technology.
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- Pastinen, T, et al.
(författare)
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Multiplex, fluorescent, solid-phase minisequencing for efficient screening of DNA sequence variation
- 1996
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Ingår i: Clinical Chemistry. - 0009-9147 .- 1530-8561. ; 42:9, s. 1391-1397
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Tidskriftsartikel (refereegranskat)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.
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