Arrayed primer extension: solid-phase four-color DNA resequencing and mutation detection technology.
The technology and application of arrayed primer extension (APEX) is presented. We describe an integrated system with DNA chip and template preparation, multiplex primer extension on the array, fluorescence imaging, and data analysis. The method is based upon an array of oligonucleotides, immobilized via the 5' end on a glass surface. A patient DNA is amplified by PCR, digested enzymatically, and annealed to the immobilized primers, which promote sites for template-dependent DNA polymerase extension reactions using four unique fluorescently labeled dideoxy nucleotides. A mutation is detected by a change in the color code of the primer sites. The technology was applied to the analysis of 10 common beta-thalassemia mutations. Nine patient DNA samples, each of which carries a different mutation, and four wild-type DNA samples were correctly identified. The signal-to-noise ratio of this technology is, on the average, 40:1, which enables the identification of heterozygous mutations with a high confidence level. The APEX method can be applied to any DNA target for efficient analysis of mutations and polymorphisms.
Kurg
A
A
Institute of Molecular and Cell Biology, Tartu Children's Hospital, University of Tartu, Estonian Biocentre.
Tõnisson
N
N
Georgiou
I
I
Shumaker
J
J
Tollett
J
J
Metspalu
A
A
D004247:DNA / Q000737:chemistry / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000295:instrumentation / Q000379:methods*; D017931:DNA Primers / Q000737:chemistry*; D005069:Evaluation Studies as Topic; D005453:Fluorescence; D005456:Fluorescent Dyes; D006580:Genetic Carrier Screening; D005820:Genetic Testing / Q000295:instrumentation / Q000379:methods; D006801:Humans; D008832:Microchemistry / Q000295:instrumentation / Q000379:methods; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017086:beta-Thalassemia / Q000175:diagnosis / Q000235:genetics*
D017931:DNA Primers; D005456:Fluorescent Dyes; D004247:DNA