Lab Med Qual Assur.  2022 Jun;44(2):105-110. 10.15263/jlmqa.2022.44.2.105.

Apolipoprotein E Genotyping Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism with General-Purpose Agarose and Lithium Borate Buffer

• Oh K
• Park G

• KMID: 2531128
• DOI: http://doi.org/10.15263/jlmqa.2022.44.2.105

Abstract

Background
Apolipoprotein E (APOE ) genotyping is an important test for predicting the risk of Alzheimer’s and cardiovascular disease. The polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) technique for APOE genotyping is not widely used due to the need for special gels. Thus, this study aimed to develop an PCR-RFLP technique to overcome this problem.
Methods
For high-resolution agarose gel electrophoresis of PCR-RFLP, a general-purpose agarose gel, lithium borate buffer (LBB), and high voltage were used. After electrophoresis, the change in the temperature of electrophoresis buffer was measured. We performed PCR-direct sequencing to confirm the results of the PCR-RFLP of genomic DNA extracted from 103 K 2 EDTA-treated venous blood samples.
Results
When a small gel 6 cm in length was run at 300 V for 50 minutes in an electrophoresis chamber with a distance of 20 cm between electrodes, the target bands could be easily discriminated and only slight deformities of the bands were observed. The change in the temperature of the buffer was 14℃. The results obtained with PCR-RFLP were in complete agreement with those of PCR-direct sequencing.
Conclusions
PCR-RFLP with electrophoresis using a general-purpose agarose gel, LBB, and high voltage is an accurate and reliable assay for APOE genotyping. Additionally, general-purpose agarose gel with LBB can be used in place of polyacrylamide or MetaPhor agarose gel for resolving small DNA fragments.

Keyword

Apolipoprotein E genotyping; Restriction fragment length polymorphism; Lithium borate buffer; Electrophoresis; Agarose