Direct fluorescence analysis of genetic polymorphisms by hybridization with oligonucleotide arrays on glass supports

Abstract

A simple and rapid method for the analysis of genetic polymorphisms has been developed using allele-specific oligonucleotide arrays bound to glass supports. Allele-specific oligonucleotides are covalently immobilized on glass slides in arrays of 3 mm spots. Genomic DNA is amplified by PCR using one fluorescently tagged primer oligonucleotide and one biotinylated primer oligonucleotide. The two complementary DNA strands are separated, the fluorescently tagged strand is hybridized to the support-bound oligonucleotide array, and the hybridization pattern is detected by fluorescence scanning. Multiple polymorphisms present in the PCR product may be detected in parallel. The effect of spacer length, surface density and hybridization conditions were evaluated, as was the relative efficacy of hybridization with single or double-stranded PCR products. The utility of the method was demonstrated in the parallel analysis of 5 point mutations from exon 4 of the human tyrosinase gene.

Influence of Dangling Ends and Surface-Proximal Tails of Targets on Probe-Target Duplex Formation in 16S rRNA Gene-Based Diagnostic Arrays

“…In one study, greater hybridization efficiency was observed with single-stranded targets than with doublestranded targets (13). This is due to competition between complementary strands and probes with double-stranded DNA.…”

Rapid detection of common Chinese glucose‐6‐phosphate dehydrogenase (G6PD) mutations by microarray‐based assay

“…First, although it is simpler to prepare double-stranded PCR products than single-stranded, hybridization of the double-stranded molecule to the chip will necessarily suffer from competition of the complementary strand with the support bound oligonucleotide probes. It was reported that hybridization efficiency on chip is much greatly with the single-stranded than the double-stranded product [5]. Second, as the longer nucleic acids form more significant secondary structure than smaller nucleic acids to affect the hybridization reaction, long nucleic acid must be fragmented then hybridized to the oligonucleotide array.…”

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.…”

“…Two main types of synthesis in situ have been described: lightbased combinatorial 16,62 or physical combination. 29,63 Deposition of pre-synthesized oligonucleotides, 20,44,64,65 cDNAs [48][49][50][51] or nucleic acids with other base and backbone modifications such as PNAs [66][67][68] have been reported. Oligonucleotide probes appear to benefit from spacer arms that link the nucleic acids to the surface.…”

“…Oligonucleotide probes appear to benefit from spacer arms that link the nucleic acids to the surface. 20,44,69 Targets have been single-or doublestranded DNA or RNA. A few investigators have explicitly examined varied hybridization conditions.…”