Computational protocol: Polymorphisms in the gene encoding bovine interleukin-10 receptor alpha are associated with Mycobacterium avium ssp. paratuberculosis infection status

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Protocol publication

[…] All SNPs were identified by sequencing PCR amplicons from each candidate gene using a DNA pool constructed with DNA from 40 Holstein bulls according to methods described in previous studies [,]. Briefly, for each bull, genomic DNA was extracted from semen and adjusted to a concentration of 5 ng/μl after several rounds of quantification using the Quant-iT PicoGreen dsDNA reagent (Invitrogen, Carlsbad, CA, USA) followed by dilution. The resultant DNA pool was amplified using the Repli-g Ultrafast mini kit (Qiagen, Santa Clara, CA, USA), and was then used as a template for PCR amplification of the 5' region and coding exons of each candidate gene. Primers were designed using Primer3 []. PCR amplicons were sequenced in both 5' and 3' orientation using an ABI Prism 3730 DNA sequencer (Applied Biosystems, Foster City CA, USA), and SNPs were identified by visual inspection of the electropherograms. Seven genes were selected for SNP discovery, IL10 [GeneId#, 281246], IL10RA [513478] and IL10RB [767864], TGFB1 [282089], TGFBR1 [282382] and TGFBR2 [535376], and SLC11A1 [282470]. Sequences were compared against GLEAN models using the Apollo Genome Annotation and Curation Tool to confirm correct gene structure (Version 1.6.5) []. In the event of a disagreement between respective GLEAN and NCBI gene models, as was the case for IL10RA, the GLEAN model was chosen. […]

Pipeline specifications

Software tools Primer3, GeneID
Application qPCR
Organisms Bos taurus, Homo sapiens
Diseases Paratuberculosis, Inflammatory Bowel Diseases, Mycobacterium avium-intracellulare Infection