PCR-RFLP genotyping protocol for chicken Mx gene G/A polymorphism associated with the S631N mutation

Area of Science:

• Molecular Biology
• Avian Genetics
• Virology

Background:

• The chicken Mx gene encodes a protein with antiviral properties.
• A specific G/A polymorphism (S631N mutation) in the Mx gene may influence antiviral activity, though its effect varies with viral strains.
• This polymorphism is potentially significant for broader viral resistance in chickens.

Purpose of the Study:

• To develop a reliable and robust Polymerase Chain Reaction - Restriction Fragment Length Polymorphism (PCR-RFLP) protocol.
• To genotype the chicken Mx gene polymorphism associated with the S631N mutation.
• To validate the developed protocol's accuracy.

Main Methods:

• Developed a PCR-RFLP assay using primers targeting the last intron and exon of the chicken Mx gene.
• Amplified a 299 bp PCR product.
• Digested PCR products with the Hpy8I restriction enzyme, differentiating between G and A alleles based on cleavage patterns.

Main Results:

• Successfully genotyped 127 chickens from three breeds (broilers, White Leghorn, New Hampshire) using the developed PCR-RFLP protocol.
• Sequencing validation confirmed 100% concordance with the PCR-RFLP results.
• The protocol demonstrated high reliability and robustness for identifying the Mx gene polymorphism.

Conclusions:

• The established PCR-RFLP protocol is a dependable method for genotyping the chicken Mx gene S631N polymorphism.
• This method can aid in studying the genetic basis of viral resistance in poultry.
• Accurate genotyping facilitates research into improving chicken immune responses to various pathogens.