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. © 1996 Wiley‐Liss, Inc.

Parallel molecular genetic analysis

“…Uses of the DNA microarray include mutation and polymorphism detection, definition of gene expression profiles, genotyping, defining gene organization and mapping, and DNA sequence analysis of previously uncharacterized regions, among others. [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] Since a high density of genes can be studied in parallel, only a small amount of sample is needed. This technology is mostly robotic driven; consequently it can easily be introduced into many research and clinical laboratories.…”

“…[40][41][42] The DNA microarray technique has been used to analyze the entire human mitochondrial DNA, 24 to detect polymorphisms in the HIV-1 clade B protease gene, 25 and to detect mutations in BRCA1, 26,36 the cystic fibrosis transmembrane receptor gene and p53. 23,34 In an extension of DNA microarray technology, Pastinen and colleagues reported allele-specific detection of 12 common disease-causing mutations in the Finnish population using a microarray and singlenucleotide extension. 43 Detection of beta-globin gene alleles and mutations has also been reported by several groups.…”

“…Detection labels have been based on fluorescence or chemiluminescence, and both primary (label on the target) and secondary (label on a molecule which binds a group on the target or duplex) detection are in use. 74 Detection devices include the proximal CCD, 64,75 CCD-microscope, 30,44 and PMT-confocal microscope [21][22][23][24][25][26] systems.…”

“…The smaller the target, the larger is D 3 and the more rapid its capture by the diffusive mechanism, supporting the empirical practice of fragmenting into smaller targets. [21][22][23][24][25][26] Fragmentation also minimizes secondary structure, an effect discussed later. H is the boundary layer, often in the length scale of tens of microns, over which the concentration changes from its bulk solution value to that right at the interface.…”

A miniature integrated device for automated multistep genetic assays

“…In a more manual format, high density GeneChip ® polynucleotide arrays have been used to analyze genetic sequences, among them the yeast genome (5) and the human mitochondrial genome (6), to characterize HIV virus polymorphisms (7), to assay RNA expression profiles (8), to detect cystic fibrosis (9) and to identify Mycobacterium tuberculosis (10) and cancerrelated mutations (11). As with most genetic analysis methods, a series of preparative reactions and processes, including enzymatic amplification and modification of a purified sample, must be carried out before hybridization analysis on the polynucleotide array.…”