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High resolution melting as an alternative method to genotype diacylglycerol O-acyltransferase 1 (DGAT1) K232A polymorphism in cattle

Author

Listed:
  • A. Abdolmohammadi

    (Department of Animal Science, Faculty of Agriculture, Razi University, Kermanshah, Iran)

  • H. Atashi

    (Department of Animal Science, Faculty of Agriculture, Shiraz University, Shiraz, Iran)

  • P. Zamani

    (Department of Animal Science, Faculty of Agriculture, Bu-Ali Sina University, Hamedan, Iran)

  • C. Bottema

    (Livestock Systems Alliance, University of Adelaide, Roseworthy, Australia)

Abstract

PCR-RFLP analysis is a common method for genotyping the DGAT1 K232A polymorphism in cattle. Our purpose was to develop a high resolution melting (HRM) assay in order to genotype the polymorphic alleles. Firstly, the PCR-RFLP method was used and the 411 bp products including the DGAT1 polymorphism were digested by CfrI enzyme. Direct sequencing was performed to confirm genotypes of the K232A polymorphism for 30 samples that presented different PCR-RFLP patterns. It was determined according to sequencing results that partial enzyme digestion had occurred for some samples. A 130 bp fragment including the polymorphism was amplified for real time PCR. Then, the HRM analysis was carried out using two fluorescent dyes, SYBR Green I and EvaGreenTM. Although the HRM genotyping using SYBR Green I was contradicted by the sequencing results, three correct melting curves were obtained for the K232A polymorphism when EvaGreenTM was used. There were no false genotypes and all genotypes were in agreement with their sequencing results. The difference in the Tm between the two homozygous groups was about 0.5°C and the AA genotypes showed a higher Tm than the KK genotypes. The heterozygous genotypes showed a different pattern. Similar results were obtained from different concentrations of EvaGreenTM in the reactions. All 206 DNA samples were genotyped using this fluorescent dye with estimated allele frequencies of 0.66 and 0.34 for the A and K alleles, respectively. Our study showed that HRM analysis will be applicable for genotyping the DGAT1 K232A polymorphism in large populations of dairy cattle.

Suggested Citation

  • A. Abdolmohammadi & H. Atashi & P. Zamani & C. Bottema, 2011. "High resolution melting as an alternative method to genotype diacylglycerol O-acyltransferase 1 (DGAT1) K232A polymorphism in cattle," Czech Journal of Animal Science, Czech Academy of Agricultural Sciences, vol. 56(8), pages 370-376.
    • Handle: RePEc:caa:jnlcjs:v:56:y:2011:i:8:id:2393-cjas
    DOI: 10.17221/2393-CJAS
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    References listed on IDEAS

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    1. E. Hradecká & J. Čítek & L. Panicke & V. Řehout & L. Hanusová, 2008. "The relation of GH1, GHR and DGAT1 polymorphisms with estimated breeding values for milk production traits of German Holstein sires," Czech Journal of Animal Science, Czech Academy of Agricultural Sciences, vol. 53(6), pages 238-246.
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    Cited by:

    1. L. Hanusová & A. Míková & L. Večerek & D. Schroeffelová & V. Řehout & L. Tothová & K. Vernerová & B. Hosnedlová & J. Čítek, 2014. "Effect of DGAT1 polymorphisms on the estimated breeding values of Czech Simmental sires," Czech Journal of Animal Science, Czech Academy of Agricultural Sciences, vol. 59(8), pages 365-373.

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    More about this item

    Keywords

    HRM analysis; PCR-RFLP; K232A polymorphism;
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