The apolipoprotein E gene (apo E) polymorphism genotyping has an allegedly important predictive value for coronary heart disorders and Alzheimer disease. Thus, interest in apo E genotyping is high for epidemiological research and this result in growing demand for simple, rapid, reliable and inexpensive technique to screen large numbers of samples.
The aim of this study was to evaluate single-tube PCR with melting temperature shift primers using real time PCR monitored by SYBR Green fluorescent dye as a rapid and inexpensive method for detection of single nucleotide polymorphism (SNP) in apo E gene compared to conventional PCR- restriction fragment length polymorphism (PCR–RFLP).
This study was conducted on 100 patients, aged from 44 to 70 years (61 ± 7.3), 28 (28%) were females and 72 (72%) were males who are previously clinically diagnosed as having coronary heart diseases and attending the Out-Patient Clinics and Inpatient Cardiology Department, Benha University hospital, , from July 2013 to March 2014.
After taking patients’ consents and under complete aseptic conditions 5ml of venous blood samples were collected, on EDTA tubes, mixed well and stored at -80 untill used.
Genomic DNA was extracted from the collected blood samples using DNA extraction kits according to manufacturer instructions. Apo E genotyping was done to detect single nucleotide polymorphism (SNP) using two different PCR-based methods:
A. PCR–RFLP (restriction fragment length polymorphism) (Haas et al., 1994; kim et al., 2010).
B. Single-tube PCR with Tm-shift primers (Papp et al., 2003; Wang et al., 2005).
In PCR-RFLP technique, the patterns of DNA fragments were determined using gel documentation system and different apo E genotypes were determined. Out of 100 patients 55% were E3/E3, 7% were E2/E3, 28% were E3/E4, 4% were E2/E4, 4% were E4/E4 and 2% were E2/E2.
The distribution of Apo E genotypes among CHD patients using single-tube PCR with Tm-shift primers were the same with the previous technique except for E2/E2=0% and E2/E3=9%. Out of 100 patients, apo E allele’s frequencies were 6.5% for Apo E ε2, 73.5% for Apo E ε3, and 20% for Apo E ε4.
In this study out of 100 DNA samples a deviation of 2% was found between the two genotyping methods (two of the samples found as E2/E2 carriers with RFLP analysis were determined as E2/E3 by single-tube with Tm shift primers procedure). The remaining 98% were all in full concordance between two methods. However, when we re-incubated the PCR products of these two cases for prolonged time with the restriction enzyme, the result corrected to become (E2/E3) and to be 100% concordance between two methods. So, the difference observed was probably due to the incomplete digestion of the amplicons in RFLP protocol.
In this study, although the cost of both methods was nearly equal, the single-tube PCR with Tm-shift primers was rapid method in comparison to RFLP technique that was time consuming.
From this study, it was concluded that:
• Apo E gene is polymorphic with three allelic variants ε2, ε3 and ε4, of which ε3 has the most common allelic frequency.
• Single-tube PCR with Tm-shift primers is simple, fast and inexpensive technique as its cost is nearly the same as PCR-RFLP. There is no risk of using radiolabelled probes. All reactions and measurements take place in a single closed tube, eliminating manipulation of PCR product and removing the risk of post-PCR contamination.
• PCR-RFLP analysis is a relatively simple technique but it is a time consuming and error-prone method due to possible incomplete restriction enzyme digestion and interpretation of the patterns often is subjective.
• There is non significant statistical difference between frequency of apo E genotypes detected by PCR-RFLP and single tube PCR in the study population.
• Tm-shift genotyping provides a diagnostic tool for apo E genotyping and medically relevant SNPs and a high-throughput SNP genotyping method for gene mapping.
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