Fast genotyping of K-RAS codons 12 and 13 based on streptavidin magnetic microbeads equipped with biotin-coupled single base-pair mismatch PCR (SM-PCR)

Fast genotyping of K-RAS codons 12 and 13 based on streptavidin magnetic microbeads equipped with biotin-coupled single base-pair mismatch PCR (SM-PCR)

  In recent years, as the public has begun to pay attention to health and preventive medicine, genetic testing has become a national development theme and academic research project that has received considerable attention. Genetic testing is to detect whether there are mutation sites in the DNA sequence in the chromosomal structure or to explore the expression level of genes. It provides the basis for the subjects and medical researchers to evaluate some genetically related diseases, physical fitness or personal characteristics. A method for precision medicine testing. At present, "precision medicine" is also actively developed, and one of which is the application of genetic testing for sequence comparison, showing the mutation status of genes, hoping to help predict the risk of disease, diagnose the disease process, analyze the prognosis and provide precise treatment, etc., and can further combine epidemiology, molecular medicine and other research to comprehensively assess the risk of suffering from diseases and health problems, and then choose the most appropriate prevention strategy or treatment plan. In recent years, Taiwan has actively developed precision medicine, also known as personalized medicine, to formulate appropriate and accurate disease prevention, diagnosis and treatment plans for individuals or specific ethnic groups based on factors such as their genetic makeup, background environment, and lifestyle.

However, the current genetic testing technology often requires the use of some expensive consumables or large-scale instruments, so not all laboratories can perform related genetic testing technology. In order to make the genetic testing technology simpler, more convenient, more accurate and faster, this research develops a "Single base pair mismatch PCR technology combined with magnetic bead gene capture platform", and applied to the rapid detection of genetic variation of codons 12 and 13 on the K-RAS gene, the detection principle is as shown in the graphic abstract. In this method, when designing primers for PCR, one base pair is deliberately designed to be mismatched with the real gene near the genetic variation. After the mismatched primer amplifies the gene, the gene sequence will contain the mismatched base pair. Then use streptavidin-modified magnetic beads to capture and purify the amplified gene fragments of the mismatched base pairs, and finally use the mismatched base pairs to match the gene variation contained in it, and design restriction enzymes to amplify only the genotype to be analyzed Fragmentation breaks down. At this time, the other end containing the FAM or TAMRA fluorescent group will be released into the liquid, so as long as the fluorescence intensity of the liquid is analyzed, the genotypes of codons 12 and 13 on the K-RAS gene can be known, and In this study, a single technique was successfully used to simultaneously analyze two genetic mutation points on codons 12 and 13 of the K-RAS gene.

    This method is convenient, simple and fast, and the method is versatile, as long as proper mismatch primers and suitable restriction enzymes are designed. It can be applied to the detection of different genes, and is quite suitable for the analysis of various types of gene variation.

Graphical Abstract 

Application and Highlights:

1. In this study, single base pair mismatch primers combined with PCR technology were used to create gene loci that can be identified by restriction enzymes, increasing the convenience of gene identification.

2. In this study, a single technique was successfully applied and two different fluorescent groups of FAM and TAMRA were used to simultaneously analyze two gene mutation points on codons 12 and 13 of the K-RAS gene.

3. This method is convenient, simple and fast, and this method is versatile. It can be applied to the detection of different genes as long as an appropriate restriction enzyme is selected, and it is quite suitable for the analysis of various types of gene variation.

Research Team Members: Tzu-Yu Pan, Hwang-Shang Kou, Shou-Mei Wu, Chun-Chi Wang

Representative Department: School of Pharmacy, College of Pharmacy, Kaohsiung Medical University

Introduction of Research Team: The team has been committed to capillary electrophoresis, pharmaceutical analysis, genetic analysis, special probe and primer design, nanoparticle synthesis and application, etc. for many years. They have developed a number of technologies for drug monitoring or genetic testing, and obtained a number of patents.

Contact Email: chunchi0716@kmu.edu.tw

Publication: Sensors & Actuators: B. Chemical, 2023, 382: 13517.

Full-Text Article: https://doi.org/10.1016/j.snb.2023.133517