Arrayed Primer Extension: Solid-Phase Four-Color DNA Resequencing and Mutation Detection Technology

Abstract

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.

Genotyping Microarray for the Detection of More Than 200 CFTR Mutations in Ethnically Diverse Populations

“…Mutations include several prominent in non-Caucasian backgrounds, including N1303K, 3849 ϩ 10 kb, 2789 ϩ 5GϾA, 3876delA, 406-1GϾA, R75X, 2055delGϾA, and S549N, which are each prevalent in the Hispanic popu- (Table 3) encompassing the mutations, with PCR mixtures that include 20% substitution of dUTPs for dTTPs, allowing for later fragmentation with uracil N-glycosylase as described previously. 4 The general applicability of a proposed diagnostic will be determined by its clinical relevance, ease of use, low cost, and number of samples that can be processed per . APEX analysis at mutation site ⌬F508.…”

“…The device combines a total internal reflection fluorescence-based excitation mechanism with a charge-coupled device camera. 4 Sequence variants were identified using Genorama 3.0 genotyping software.…”

“…It is an integrated system with DNA microarray chip, multiplex primer extension on the array, and fluorescence imaging and data analysis. 4,14 Because of the unprecedented number and selection of mutations on the CFTR array, this CF test has a higher mutation detection capability than assays currently available on-site in diagnostic laboratories. It also more than doubles the number of offered mutations on currently available panels at reference laboratories.…”

“…In a one-step reaction, the functional inactivation of the traces of unincorporated dNTPs was achieved by addition of shrimp alkaline phosphatase (Amersham Pharmacia Biotech, Inc., Milwaukee, WI), and fragmentation of the PCR product was achieved by the addition of thermolabile uracil N-glycosylase (Epicenter Technologies, Madison, WI) followed by heat treatment. 4 …”

“…The assay is based on singleprimer nucleotide extension, first described by Shumaker et al 5 in 1996 and subsequently converted to an array format. 4 It is a methodology that enables accurate mutation detection in a microarray format. Our CF APEX microarray described herein is suitable for carrier screening as well as molecular diagnosis of affected individuals.…”