The overall goal of this method is to genotype complement receptor 1 length polymorphisms for use in the assessment of susceptibility to diseases, such as Alzheimer's disease, to better understand the role of CR1 isoforms in disease pathogenesis. This method can help answer key questions in the neuroscience field such as the pathogenesis of Alzheimer's disease. The main advantages of this technique is that it does not require fresh blood samples and it's easier, cheaper, faster and therefore applicable to larger populations.
We first had the idea for this method when we recognize the challenges in performing Western blots regarding the necessary time and need for fresh blood samples. Pipette 20 microliters of proteinase K into the bottom of a 1.5-milliliter tube. Then, add 200 microliters of sample to the tube.
Add 200 microliters of lysis buffer to the sample. Mix by pulse vortexing for 15 seconds. After mixing, incubate at 56 degrees Celsius for 10 minutes in a water bath.
Briefly centrifuge the 1.5-milliliter tube to remove the drops from the inside of the lid. Then, add 100 microliters of ethanol to the sample. Mix again by pulse vortexing for 15 seconds before briefly centrifuging the tube as before.
Next, carefully apply the mixture to the membrane column in a two-milliliter collection tube. Without wetting the rim, close the cap and centrifuge at 6, 000 times G for one minute. Place the membrane column in a clean two-milliliter collection tube and discard the tube containing the filtrate.
Carefully open the membrane column and add 500 microliters of wash buffer one without wetting the rim. Close the cap and centrifuge at 6, 000 times G for one minute. Then, place the membrane column in a clean two-milliliter collection tube and discard the tube containing the filtrate.
After carefully opening the membrane column, add 500 microliters of wash buffer two without wetting the rim. Close the cap and centrifuge at 20, 000 times G for three minutes. Then, place the membrane column in a new two-milliliter collection tube and centrifuge again.
Following centrifugation, place the membrane column in a clean 1.5 milliliter tube and discard the collection tube containing the filtrate. Carefully open the membrane column and add 200 microliters of distilled water. Incubate the column at room temperature for five minutes before centrifuging at 6, 000 times G for one minute.
To perform the HRM-PCR protocol, first dilute the DNA samples in 1.5-milliliter tubes with water to a concentration of 10 nanograms per microliter. Next, dilute the thawed primer solutions in 1.5-milliliter tubes with water to the same concentration of six micromolar. Thaw the HRM-PCR kit solutions and mix carefully by vortexing to ensure the recovery of all contents.
Briefly spin the three vials containing enzymatic mixture with DNA binding dye, magnesium chloride and water in a microcentrifuge before opening them. In a 1.5-milliliter tube at room temperature, use the components to prepare the PCR mix for 120-microliter reaction as described in the text protocol. Mix carefully by vortexing.
It is critical that you work with attention and application according to good practice in molecular biology. Pipette 19 microliters of the PCR mix into each well of a white multi-well plate. Add one microliter of the concentration-adjusted DNA template, then seal the white multi-well plate with sealing foil.
Centrifuge the white multi-well plate for one minute at 1, 500 times G in a standard swing-bucket centrifuge containing a rotor for multi-well plates with suitable adapters. Load the white multi-well plate into the HRM-PCR instrument. Start the HRM-PCR program with the PCR conditions found in the text protocol.
Following the HRM-PCR reaction, perform HRM analysis to determine the CR1 length polymorphism as described in the text protocol. Fusion curves were analyzed using the software after the high-resolution melting of the PCR-derived amplicons from the genomic DNA of subjects with different CR1 isoforms. The results are curves that discriminate subjects according to their allotypic CR1 expression phenotypes.
As shown by the two modes of presentation, normalized and shifted melting curves and normalized and temp-shifted difference plot, the curve profiles are distributed according to the CR1 star three, CR1 star one group, the CR1 star one, CR1 star two group, the CR1 star one group, the CR1 star two group and the CR1 star two, CR1 star four group. These curves correspond to the CR1 length polymorphism alleles. After watching this video, you should have a good understanding of how to easily genotype the CR1 length polymorphism according to perfect curves obtained by HRM-PCR.
Generally, individuals new to this method will struggle because of the need of reference samples of already known CR1 length genotypes. When mastered, this technique can be done in four hours if it is performed properly. While attempting this procedure, it's important to remember to use the same concentration for each DNA sample.
Following this procedure, other methods like conical DNA sequencing can be performed in order to answer additional questions like the finding of a new known private SNP. After this development, this technique paves a way for researches in the field of population genetics to explore the selective pressure of pathogens in CR1 length polymorphism. Though this method can provide insight into susceptibility in pathogenesis of Alzheimer's disease, it can also be applied to other study of disease such as systemic lupus erythematosus and malaria.
