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High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer's Disease Gene Susceptibility Assessment.
- Source :
-
Journal of visualized experiments : JoVE [J Vis Exp] 2017 Jul 18 (125). Date of Electronic Publication: 2017 Jul 18. - Publication Year :
- 2017
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Abstract
- Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but especially on red blood cells (RBCs). As a receptor for the complement components C3b and C4b, CR1 regulates the activation of the complement cascade and promotes the phagocytosis of immune complexes and cellular debris, as well as the amyloid-beta (Aβ) peptide in Alzheimer's disease (AD). Several studies have confirmed AD-associated single nucleotide polymorphisms (SNPs), as well as a copy-number variation (CNV) in the CR1 gene. Here, we describe an innovative method for determining the length polymorphism of the CR1 receptor. The receptor includes three domains, called long homologous repeats (LHR)-LHR-A, LHR-C, and LHR-D-and an n domain, LHR-B, where n is an integer between 0 and 3. Using a single pair of specific primers, the genetic material is used to amplify a first fragment of the LHR-B domain (the variant amplicon B) and a second fragment of the LHR-C domain (the invariant amplicon). The variant amplicon B and the invariant amplicon display differences at five nucleotides outside of the hybridization areas of said primers. The numbers of variant amplicons B and of invariant amplicons is deduced using a quantitative tool (high-resolution melting (HRM) curves), and the ratio of the variant amplicon B to the invariant amplicon differs according to the CR1 length polymorphism. This method provides several advantages over the canonical phenotype method, as it does not require fresh material and is cheaper, faster, and therefore applicable to larger populations. Thus, the use of this method should be helpful to better understand the role of CR1 isoforms in the pathogenesis of diseases such as AD.
- Subjects :
- Alzheimer Disease genetics
DNA isolation & purification
DNA metabolism
Disease Susceptibility
Erythrocytes metabolism
Genotype
Humans
Phase Transition
Phenotype
Polymorphism, Single Nucleotide
Protein Isoforms genetics
Protein Isoforms metabolism
Receptors, Complement metabolism
Software
Video Recording
Alzheimer Disease pathology
Amplified Fragment Length Polymorphism Analysis methods
Receptors, Complement genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1940-087X
- Issue :
- 125
- Database :
- MEDLINE
- Journal :
- Journal of visualized experiments : JoVE
- Publication Type :
- Academic Journal
- Accession number :
- 28745649
- Full Text :
- https://doi.org/10.3791/56012