Effect of oligonucleotide probes substituted by deoxyinosines on the specificity of SNP detection on the DNA microarray

Abstract

One of the main factors that can affect the quality of microarray results is the microarray hybridization specificity. The key factor that affects hybridization specificity is the design of the probes. In this paper, we described a novel oligonucleotide probe containing deoxyinosines aimed at improving DNA hybridization specificity. We compared different probes to determine the distance between deoxyinosine base and SNPs site and the number of deoxyinosine bases. The new probe sequences contained two set of deoxyinosines (each set had two deoxyinosines), in which the interval between SNP site and each set of deoxyinosines was two bases. The new probes could obtain the highest hybridization specificity. The experimental results showed that probes containing deoxyinosines hybridized effectively to the perfectly matched target and improved the hybridization specificity of DNA microarray. By including a simple washing step after hybridization, these probes could distinguish matched targets from single-base-mismatched sequences perfectly. For the probes containing deoxyinosines, the fluorescence intensity of a match sequence was more than eight times stronger than that of a mismatch. However, the intensity ratio was only 1.3 times or less for the probes without deoxyinosines. Finally, using hybridization of the PCR product microarrays, we successfully genotyped SNP of 140 samples using these new labeled probes. Our results show that this is a useful new strategy for modifying oligonucleotide probes for use in DNA microarray analysis.

Rapid and Unamplified Detection of SARS-CoV-2 RNA via CRISPR-Cas13a-Modified Solution-Gated Graphene Transistors

“…6,13 For nucleic acid biosensors based on the complementary base pairing rule, the specificity of hybridization is one of the main factors affecting the detection results. 14 For some oligonucleotide probes that are simply immobilized on the sensing surface, thermodynamic equilibrium conditions cannot be obtained without sufficient incubation time. As a result, the hybridization may be kinetically or sterically inaccessible.…”