Secure and optimized detection of PNPLA3 rs738409 genotype by an improved PCR-restriction fragment length polymorphism method
- The PNPLA3 reference single-nucleotide polymorphism rs738409 has been identified as a predisposing factor for nonalcoholic fatty liver disease. A simple method based on PCR and restriction fragment length polymorphism (RFLP) analysis had been published to detect the nonpathogenic allele PNPLA3 rs738409 variant. The presence of the pathogenic variant was deduced by the indigestibility of the corresponding PCR product with BtsCI recognizing the nonpathogenic allele. However, one cannot exclude that an enzymatic reaction does not occur for other, more trivial, reasons. For safe and secure detection of the pathogenic PNPLA3 rs738409, we have further developed the PCR-restriction fragment length polymorphism method by adding a second restriction enzyme digest, clearly identifying the correct PNPLA3 alleles and in particular the pathogenic variant. <br /> METHOD SUMMARY <br /> The method presented here represents an improved genetic diagnosis of the PNPLA3 rs738409 alleles based on conventional and inexpensive molecular biological methods.The PNPLA3 reference single-nucleotide polymorphism rs738409 has been identified as a predisposing factor for nonalcoholic fatty liver disease. A simple method based on PCR and restriction fragment length polymorphism (RFLP) analysis had been published to detect the nonpathogenic allele PNPLA3 rs738409 variant. The presence of the pathogenic variant was deduced by the indigestibility of the corresponding PCR product with BtsCI recognizing the nonpathogenic allele. However, one cannot exclude that an enzymatic reaction does not occur for other, more trivial, reasons. For safe and secure detection of the pathogenic PNPLA3 rs738409, we have further developed the PCR-restriction fragment length polymorphism method by adding a second restriction enzyme digest, clearly identifying the correct PNPLA3 alleles and in particular the pathogenic variant. <br /> METHOD SUMMARY <br /> The method presented here represents an improved genetic diagnosis of the PNPLA3 rs738409 alleles based on conventional and inexpensive molecular biological methods. We used methodology based on PCR and restriction fragment length polymorphisms and clearly identified both described alleles by the use of two restriction enzymes. Digestion of individuals' specific PNPLA3 PCR fragments with both enzymes in independent reactions clearly showed the PNPLA3 rs738409 genotype.…
Author details: | Jörg EnssleORCiD, Karsten-Henrich WeylandtORCiDGND |
---|---|
DOI: | https://doi.org/10.2144/btn-2020-0163 |
ISSN: | 0736-6205 |
ISSN: | 1940-9818 |
Pubmed ID: | https://pubmed.ncbi.nlm.nih.gov/33956487 |
Title of parent work (English): | BioTechniques : the international journal of life science methods |
Publisher: | Future Science Ltd. |
Place of publishing: | London |
Publication type: | Article |
Language: | English |
Date of first publication: | 2021/05/06 |
Publication year: | 2021 |
Release date: | 2023/12/08 |
Tag: | PCR– RFLP; PNPLA3; disease; n-3 polyunsaturated fatty acid therapies; nonalcoholic fatty liver; rs738409 |
Volume: | 70 |
Issue: | 6 |
Number of pages: | 5 |
First page: | 345 |
Last Page: | 349 |
Funding institution: | Faculty of Health Sciences, Faculty of the Brandenburg University of Technology Cottbus -Senftenberg; Brandenburg Medical School Theodor Fontane; University of Potsdam; MHB Open Access Publication Fund - the German Research Association (DFG)German Research Foundation (DFG) |
Organizational units: | Fakultät für Gesundheitswissenschaften |
DDC classification: | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
6 Technik, Medizin, angewandte Wissenschaften / 60 Technik / 600 Technik, Technologie | |
6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit | |
Peer review: | Referiert |
Publishing method: | Open Access / Gold Open-Access |
DOAJ gelistet | |
License (German): | CC-BY-NC-ND - Namensnennung, nicht kommerziell, keine Bearbeitungen 4.0 International |