Computational protocol: Single nucleotide polymorphism mining and nucleotide sequence analysis of Mx1 gene in exonic regions of Japanese quail

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Protocol publication

[…] The single nucleotide polymorphisms (SNPs) of Mx1 gene were identified by PCR-SSCP technique [,]. The PCR products were resolved on 15% polyacrylamide gel. About 6 µl of PCR product and 12 µl of denaturing formamide dye (formamide, 95%; xylene cyanol, 0.025%; bromophenol blue, 0.025%; 0.5 M EDTA, 4%) were taken in a 0.2 ml PCR tube and mixed properly. The mixture of PCR product and formamide dye were denatured at 95°C for 10 min (by keeping in hot water bath) and snap chilled on ice for 15 min. The product was loaded in gel carefully. The electrophoresis was performed at 4°C for 13-16 h at 130 constant volts. For visualization of bands, silver staining was carried out. The pattern of DNA bands were documented by gel documentation system. The genotypes were identified, and the different SNPs were scored on banding pattern of SSCP. The gene and genotype frequencies were estimated []. The identified genotypes were custom sequenced and analyzed by BLAST (www.ncbi.nlm.nih.gov/BLAST). The nucleotide sequences and chromatograms were aligned and evaluated using BioEdit v7.0.5 []. The phylogenetic trees were constructed using MEGA 6 []. […]

Pipeline specifications

Software tools BioEdit, MEGA
Application Phylogenetics
Organisms Coturnix japonica, Gallus gallus
Chemicals Nucleotides