Determination of the six ApoE genotypes using an isolated probe-polymerase chain reaction technique combined with multiplexed unlabeled probes and high resolution melting analysis

Apolipoprotein E (ApoE) is a polymorphic glycoprotein consisting of 299 amino acids. ApoE is responsible for 5-15% of the total cholesterol level in the body and is strongly associated with coronary heart disease and specifically Type III hyperlipidemia. ApoE is also linked to Alzheimer's disease (AD). The ApoE gene contains two single nucleotide polymorphisms (SNPs at amino acid positions 112 and 158 giving rise to 3 homozygous and 3 heterozygous genotypes for total of 6 different genotypes. ApoE has been the subject of much study since it was discovered in the early 1970s using a gel separation process called isoelectric focusing. Diagnostic testing for SNPs has continually evolved through the decades as new technologies emerge. One such advance was elucidation of the polymerase chain reaction. The polymerase chain reaction (PCR) has revolutionized the ability to examine DNA at the molecular level. The theoretical simplicity of the PCR has led to an ever expanding array of techniques and uses for PCR. One of these techniques is asymmetric PCR. Asymmetric PCR is used to selectively amplify on strand of the genomic DNA. Isolated probe-polymerase chain reaction (IP-PCR) is a new method that expands the usefulness of asymmetric PCR and unlabeled probes. IP-PCR separates the asymmetric PCR amplification and the detection of the unlabeled probes while maintaining a closed tub environment. This prevents the probes from interfering with the amplification of the DNA target. The closed tub format eliminates the possibility of amplicon contamination or sample carryover. To test this new technique DNA was isolated from 101-de-identified blood samples, obtained from ARUP. The DNA from these samples was genotyped using the IP-PCR technique using multiplexed unlabeled probes and high resolution melting analysis. The results from ARUP were de-blinded and compared to results obtained by IP-PCR. The IP-PCR results agreed in 96 out of 101 samples. The 5 discordant samples did not display any abnormal melting curve plots when compared to other samples tested. These 5 discordant samples were sequenced to determine the correct ApoE genotype. The sequencing results showed that IP-PCR and high resolution melting using unlabeled probes gave correct genotype in all five cases. Two samples (sample 20 and 36) out of the 101 samples displayed abnormal melting curve plots. However the abnormalities did not interfere with determining the genotype of these two samples. The 2 samples were also sequenced. The sequencing results showed no mutation under the unlabeled probe. The IPCR technique correctly genotyped every DNA sample use in the experiment.