PUBLICATION
A Simple, Robust, and Cost-effective Method for Genotyping Small-scale Mutations
- Authors
- Zheng, L., Hill, J., Zheng, L., Rumi, M.A.K., Zheng, X.L.
- ID
- ZDB-PUB-221025-10
- Date
- 2022
- Source
- Journal of clinical and translational pathology 2: 108-115 (Journal)
- Registered Authors
- Zheng, Liang
- Keywords
- Agarose gel electrophoresis, Gene editing, Genotyping, Polymerase chain reaction, T7E1 endonuclease
- MeSH Terms
- none
- PubMed
- 36276172 Full text @ J Clin Transl Pathol
Citation
Zheng, L., Hill, J., Zheng, L., Rumi, M.A.K., Zheng, X.L. (2022) A Simple, Robust, and Cost-effective Method for Genotyping Small-scale Mutations. Journal of clinical and translational pathology. 2:108-115.
Abstract
Background and objectives Genotyping is an important tool for studying gene functions in animals or detecting genetic variants in humans. Various methods using low to high concentrations of agarose or polyacrylamide gel electrophoresis have been developed for genotyping. These methods rely on the detection of large-size differences (20-2,000 bp) of targeted PCR products between a wild-type gene and a mutant gene. Endonuclease digestion was introduced to identify heterozygous mutations, but it was not possible to differentiate the wild-type from the homozygous mutants with the same or similar size. This study thus developed a novel, simple, and reliable test for genotyping animals or cells following genetic modifications.
Methods We developed an improved and simple method that used 2% agarose gel electrophoresis following T7E1 or Surveyor endonuclease digestion to firstly separate the heterozygous mutations from the wild-type or homozygous mutations. By adding a wild-type PCR product to a potentially homozygous product, which would form heteroduplexes, we could then separate the wild-type from a homozygous mutation with a nearly identical size or only a single base pair substitution without Sanger sequencing.
Results We verified this method in genotyping zebrafish mutants with a 2-8-bp deletion or insertion and mouse mutants with a 1- or 8-bp substitution. The wild-type, heterozygous, and homozygous mutations ranged 1-8 bp were clearly differentiated on agarose gel. Sanger sequencing also confirmed our genotyping results.
Conclusions This novel and improved genotyping method may have a broad application in many clinical and research laboratories for rapid and economical genotyping of patients and animals with a small area deletion or single base pair substitution, particularly in the era of gene editing or in those with naturally occurring mutations.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping