Fluorescence detection of apolipoprotein E gene polymorphisms based on oligonucleotide ligation and magnetic separation

• Published in: Analytical methods Vol. 15; no. 36; pp. 471 - 4717
• Main Authors: Wen, Wan-Chen, Lin, Yi-Hui, Duh, Tsai-Hui, Chen, Chun-Hsien, Feng, Chia-Hsien, Chen, Yen-Ling
• Format: Journal Article
• Language: English
• Published: Cambridge Royal Society of Chemistry 21.09.2023
• Subjects: Alleles; Alzheimer's disease; Apolipoprotein E; Apolipoproteins; Biotin; Cell death; Dementia disorders; Deoxyribonucleic acid; DNA; DNA probes; E protein; Fluorescence; Fluorescent indicators; Gene polymorphism; Gene sequencing; Genotypes; Health risks; Magnetic separation; Neurodegenerative diseases; Nucleotide sequence; Nucleotides; Oligonucleotides; Single-nucleotide polymorphism; Streptavidin; Target detection
• ISSN: 1759-9660; 1759-9679
• DOI: 10.1039/d3ay01245j

Alzheimer's disease is a progressive neurodegenerative condition that causes brain cell death and is the leading cause of dementia. Most patients with Alzheimer's disease are diagnosed with late-onset Alzheimer's disease (LOAD), with apolipoprotein E (APOE) genotypes being highly associated with the frequency of LOAD risk. A fluorescence detection system coupled with oligonucleotide ligation and magnetic separation was developed to identify two single-nucleotide polymorphisms (SNPs) for the APOE gene and recognize APOE alleles for LOAD. The system utilized a fluorescence probe with one base-discriminating nucleoside for SNP (F probe) and a perfectly complementary biotin-modified sequence against the target DNA (P probe). When the F and P probes matched the target DNA sequences, DNA ligation occurred, and ligation products were produced. Streptavidin magnetic beads were subsequently employed to remove the ligation products, and a decrease in fluorescence intensity was observed in the supernatant compared to when there was no target DNA. This system detected two SNPs of APOE alleles, namely rs429358 and rs7412. The results indicated that the R -values (( F 0 − F 1 )/ F 0 ) for rs429358 were 0.92 ± 0.002 for the T/T target, 0.47 ± 0.004 for the T/C target and 0.11 ± 0.004 for the C/C target, respectively. The R -values for rs7412 were 0.73 ± 0.009 for the C/C target, 0.42 0.001 for the C/T target and 0.16 ± 0.007 for the T/T target, respectively. F 0 and F 1 represent the fluorescence intensity of the F probe without and with target DNA, respectively. Based on fluorescence intensity, the fluorescence detection system was able to identify the genotypes of the APOE gene accurately to evaluate the risk of Alzheimer's disease. A fluorescence detection system coupled with oligonucleotide ligation and magnetic separation was developed to identify two single-nucleotide polymorphisms for the APOE gene.