Genscanner and Lightscanner32 non-labeled probe method genotyping
Apolipoprotein (ApoE) Multiple Experiment

Codon 112 (T>C)
Codon 158 (C>T)

Experimental background

      Apolipoprotein (ApoE) is a glycoprotein, including 299 amino acids, that plays a major role in regulating cholesterol levels in the body. Excessive cholesterol levels are a major cause of coronary heart disease. In the United States, coronary heart disease is a major cause of death. ApoE is also associated with Alzheimer's disease, which is caused by atrophy of the frontal lobe of the brain, causing neurasthenia. The common manifestation of Alzheimer's disease is dementia, and it cannot be cured, degraded, and terminal. The ApoE gene has two SNPs located at codon 112 (c.388 T>C) and 158 (c.526 C>T). The genotypes of codons 112 and 158 combine to translate three different types of proteins, commonly referred to as: ε3 (normal), ε2 (abnormal), and ε4 (abnormal).


• The ε2 allele is linked to the high-lipoproteinemia genetic defect type III, increasing or decreasing the risk of arteriosclerosis.
• In the population, approximately 64% of people have the ε3 allele, which is considered a “neutral” ApoE genotype.
• The ε4 allele is associated with arteriosclerosis and Alzheimer's disease, impairing cognitive function and reducing neurite outgrowth.

method

      Similar PCR methods for genotyping generally require fluorescently labeled nucleotide probes. We use a 3'-end, unlabeled probe (Luna Probes) and LC Green saturated dye to generate a unique melting curve for the genotype under the probe. The key to this approach is the unbalanced ratio of PCR primers, which results in asymmetric PCR or overproduction of a single strand (the target strand of the probe in the experiment) so that sufficient signal can be generated.
The following reagents were required to establish a successful Lightscanner instrument.

Prepare samples

      The High Resolution Melting Curve (HRM) compares the melting map of the independent PCR reactions, so it is critical to minimize the variation between reactions. Samples to be analyzed with HRM must be extracted using the same extraction platform. This eliminates minor differences in the composition of the reagents between the last eluting buffers of different extraction platforms. For the HRM experiment, it is recommended that the content of the 10 ul system genomic DNA template is 10-20 ng.

experimental design

Note: The underlined red font shows the position of the SNP locus in the unlabeled probe. The "block" shows a C3 closure that prevents the 3' end of the probe from extending. Based on the small features of the 112 probe, it was designed to mismatch with the target strand to alter the Tm value of the probe. This allows for differences in the temperatures of the 112 and 158 probes in the same reaction.

reaction system