Allele-specific polymerase chain reaction for the detection of Alzheimer's disease-related single nucleotide polymorphisms

Abstract

Background: The incidence of Alzheimer's disease, particularly in developing countries, is expected to increase exponentially as the population ages. Continuing research in this area is essential in order to better understand this disease and develop strategies for treatment and prevention. Genome-wide association studies have identified several loci as genetic risk factors of AD aside from apolipoprotein E such as bridging integrator (BIN1), clusterin (CLU), ATP-binding cassette sub-family A member 7 (ABCA7), complement receptor 1 (CR1) and phosphatidylinositol binding clathrin assembly protein (PICALM). However genetic research in developing countries is often limited by lack of funding and expertise. This study therefore developed and validated a simple, cost effective polymerase chain reaction based technique to determine these single nucleotide polymorphisms.

Methods: An allele-specific PCR method was developed to detect single nucleotide polymorphisms of BIN1 rs744373, CLU rs11136000, ABCA7 rs3764650, CR1 rs3818361 and PICALM rs3851179 in human DNA samples. Allele-specific primers were designed by using appropriate software to permit the PCR amplification only if the nucleotide at the 3'-end of the primer complemented the base at the wild-type or variant-type DNA sample. The primers were then searched for uniqueness using the Basic Local Alignment Search Tool search engine.

Results: The assay was tested on a hundred samples and accurately detected the homozygous wild-type, homozygous variant-type and heterozygous of each SNP. Validation was by direct DNA sequencing.

Conclusion: This method will enable researchers to carry out genetic polymorphism studies for genetic risk factors associated with late-onset Alzheimer's disease (BIN1, CLU, ABCA7, CR1 and PICALM) without the use of expensive instrumentation and reagents.

Figure 1

The electrophoresis profiles for some of the successful amplifications.BIN1 rs744373 (A), CLU rs11136000 (B), ABCA7 rs3764650 (C), CR1 rs3818361 (D) and PICALM rs3851179 (E). W = lane for wild-type amplification, V = lane for variant-type amplification.

Figure 2

Illustrations of PCR amplification and size of fragments. The common primers (A) flank and amplify the non-specific-allele amplicons while the allele-specific primers (B) amplify the specific-allele amplicons to allow the differentiation of the genotypes. The DNA sequences were retrieved from GenBank (National Center for Biotechnology) using accession numbers (C). The non-specific-allele amplicons act as internal positive control (E). dsDNA = double-stranded DNA, Wt = wild-type, Vt = variant-type.