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Quantification of allelic differential expression using a simple Fluorescence primer PCR-RFLP-based method.

ABSTRACT: Allelic differential expression (ADE) is common in diploid organisms, and is often the key reason for specific phenotype variations. Thus, ADE detection is important for identification of major genes and causal mutations. To date, sensitive and simple methods to detect ADE are still lacking. In this study, we have developed an accurate, simple, and sensitive method, named fluorescence primer PCR-RFLP quantitative method (fPCR-RFLP), for ADE analysis. This method involves two rounds of PCR amplification using a pair of primers, one of which is double-labeled with an overhang 6-FAM. The two alleles are then separated by RFLP and quantified by fluorescence density. fPCR-RFLP could precisely distinguish ADE cross a range of 1- to 32-fold differences. Using this method, we verified PLAG1 and KIT, two candidate genes related to growth rate and immune response traits of pigs, to be ADE both at different developmental stages and in different tissues. Our data demonstrates that fPCR-RFLP is an accurate and sensitive method for detecting ADE on both DNA and RNA level. Therefore, this powerful tool provides a way to analyze mutations that cause ADE.

SUBMITTER: Zhao C

PROVIDER: S-EPMC6474871 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Quantification of allelic differential expression using a simple Fluorescence primer PCR-RFLP-based method.

Zhao Changzhi C Xie Shengsong S Wu Hui H Luan Yu Y Hu Suqin S Ni Juan J Lin Ruiyi R Zhao Shuhong S Zhang Dingxiao D Li Xinyun X

Scientific reports 20190419 1

Allelic differential expression (ADE) is common in diploid organisms, and is often the key reason for specific phenotype variations. Thus, ADE detection is important for identification of major genes and causal mutations. To date, sensitive and simple methods to detect ADE are still lacking. In this study, we have developed an accurate, simple, and sensitive method, named fluorescence primer PCR-RFLP quantitative method (fPCR-RFLP), for ADE analysis. This method involves two rounds of PCR amplif ...[more]

PMID: 31004110

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Project description:BackgroundGenotyping of polymorphic chromosomal inversions in malaria vectors such as An. coluzzii Coetzee & Wilkerson is important, both because they cause cryptic population structure that can mislead vector analysis and control and because they influence epidemiologically relevant eco-phenotypes. The conventional cytogenetic method of genotyping is an impediment because it is labor intensive, requires specialized training, and can be applied only to one gender and developmental stage. Here, we circumvent these limitations by developing a simple and rapid molecular method of genotyping inversion 2Rc in An. coluzzii that is both economical and field-friendly. This inversion is strongly implicated in temporal and spatial adaptations to climatic and ecological variation, particularly aridity.MethodsUsing a set of tag single-nucleotide polymorphisms (SNPs) strongly correlated with inversion orientation, we identified those that overlapped restriction enzyme recognition sites and developed four polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) assays that distinguish alternative allelic states at the tag SNPs. We assessed the performance of these assays using mosquito population samples from Burkina Faso that had been cytogenetically karyotyped as well as genotyped, using two complementary high-throughput molecular methods based on tag SNPs. Further validation was performed using mosquito population samples from additional West African (Benin, Mali, Senegal) and Central African (Cameroon) countries.ResultsOf four assays tested, two were concordant with the 2Rc cytogenetic karyotype > 90% of the time in all samples. We recommend that these two assays be employed in tandem for reliable genotyping. By accepting only those genotypic assignments where both assays agree, > 99% of assignments are expected to be accurate.ConclusionsWe have developed tandem PCR-RFLP assays for the accurate genotyping of inversion 2Rc in An. coluzzii. Because this approach is simple, inexpensive, and requires only basic molecular biology equipment, it is widely accessible. These provide a crucial tool for probing the molecular basis of eco-phenotypes relevant to malaria epidemiology and vector control.

Dataset Information

Quantification of allelic differential expression using a simple Fluorescence primer PCR-RFLP-based method.

Publications

Quantification of allelic differential expression using a simple Fluorescence primer PCR-RFLP-based method.

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