Cystic fibrosis mutation detection by hybridization to light‐generated DNA probe arrays

Abstract

We have combined photochemistry and photolithography with solid-phase DNA synthesis chemistry to form a new technology that makes high density oligonucleotide probe array synthesis possible. Hybridization to these two-dimensional arrays containing hundreds or thousands of oligonucleotide probes provides a powerful DNA sequence analysis tool. Two types of light-generated DNA probe arrays have been used to test for a variety of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. One array, made up of 428 probes, was designed to scan through the length of CFTR exon 11 and identify differences from the wild type reference sequence. The second type of array contained 1480 probes chosen to detect known deletions, insertions, or base substitution mutations. The validity of the probe arrays was established by hybridizing them with fluorescently labeled control oligonucleotide targets. Characterized mutant CFTR genomic DNA samples were then used to further test probe array hybridization specificity. Finally, ten unknown patient samples were genotyped using the CFTR probe array assay. The genotype assignments were identical to those obtained by PCR product restriction fragment analysis. Our results show that light-generated DNA probe arrays are highly effective in analyzing complex mutation and polymorphism patterns in a relatively large gene such as CFTR.

Microarray analysis in cystic fibrosis

“…While many of these mutations are extremely rare and not all mutations are disease-causing, there is a clear need for a novel platform for genetic testing of CF which can provide simultaneous screening of a large proportion of CFTR mutations with a high level of accuracy and precision, affordable cost, and which would be compatible with a high throughput screening programme [5]. DNA microarray platforms have been developed using different approaches to implement mutation detection [3,6,7], and thus the potential exists for such platforms to provide a complete solution to CFTR mutation detection for CF screening.…”

Genome-wide mapping with biallelic markers in Arabidopsis thaliana

“…Of 48 markers that did not display discrimination, 45 (94%) were shown to have been sequenced correctly, confirming that failure of markers is attributable to lack of discrimination by the array. It has been shown that more extensive optimization of conditions and array design will increase the percentage of functional markers; however, sufficient markers displayed discrimination for the purposes of this study 18 .…”

Two-dye based arrayed primer extension for simultaneous multigene detection in lipid metabolism

“…Replacement of 32% dTTPs with dUTP, which generated DNA templates of 50-150 bp, was optimal in this APEX study. Most studies have used 20% dUTP [25][26][27][28][29]. There was only one study that used 15% dUTP [37] in the PCR reaction.…”

“…Before adding to the APEX reaction, the PCR products were digested into smaller fragments using uracil Nglycosylase and heat treatment. This enzyme specifically hydrolyzes the N-glycosilic bond connecting uracil to the deoxyribose sugar and generates abasic sites in DNA [25]. After digestion, 100-300 bp and Table 3 List of forty-eight genotypes from eight DNA samples detectable with the APEX microarray.…”

“…Previous studies have shown that the maximum length of DNA template that can be used is 200 bp, but 100 bp is optimal [23]. A longer PCR product should be fragmented by replacing a fraction of dTTPs with dUTPs in the PCR reaction and the uracil bases digested with uracil-N-glycosylase [25]. Replacement of 32% dTTPs with dUTP, which generated DNA templates of 50-150 bp, was optimal in this APEX study.…”