RT Journal
A1 Pastinen, Tomi
A1 Kurg, Ants
A1 Metspalu, Andres
A1 Peltonen, Leena
A1 Syvänen, Ann-Christine
T1 Minisequencing: A Specific Tool for DNA Analysis and Diagnostics on Oligonucleotide Arrays
JF Genome Research 
JO Genome Research 
YR 1997 
FD June 01 
VO 7 
IS 6 
SP 606 
OP 614 
DO 10.1101/gr.7.6.606 
UL http://genome.cshlp.org/content/7/6/606.abstract 
AB 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.