ORIGINAL ARTICLE

High resolution melting analysis on temperature allows the genotype determination of indicators related to beef

El análisis de alta resolución en la temperatura de disociación permite genotipificación de marcadores relacionados con la carne de bovino

L. E. López Rojas,^I,II Laura Patiño Cadavid,^I Juliana María Martínez Garro,^I Silvana Durán Ortiz,^I Lina Johana Correa Agudelo,^I Santiago González Escudero,^I Albeiro López Herrera,^II José Julián Echeverri Zuluaga,^II

ABSTRACT

Some SNPs within µ-calpain (CAPN1), calpastatin (CAST) and leptin (LEP) genes have been associated to beef tenderness and, in order to optimize the processes of genetic improvement in cattle rearing farms, it is necessary to apply fast, reliable and accessible methodologies to producers, for determining the genotype of these polymorphisms. Although there are several methodologies, the HRM is stated as a promising alternative that meets these conditions. Therefore, the PCR-HRM was standardized and validated for genotyping the SNPs CAPN4751, CAPN316, CAST2959, CAST282, E2FB and E2JW of 50 animals from Bos taurus and Bos indicus breeds, using PCR-RFLP as gold standard test. There was an agreement among the results obtained by PCR-HRM, compared to PCR-RFLP, and high sensitivity, specificity and appropriate negative and positive predictive values for PCR-HRM.  Similarly, repeatability and reproducibility were verified after obtaining homogeneity in the intra-run and inter-run results. Although the results show that the PCR-HRM is an efficient technique, and allows to obtain reliable results in a short time, these can be affected by the extraction method, quality and quantity of DNA, among other undetermined factors that can generate false positives and atypical dissociation profiles. Therefore, these variables must be controlled and the PCR should be monitored to identify unusual dissociation curves, caused by excess or deficiency in the amplification.

Key words: quality, calpastatin, leptin, µ-calpain, HRM.

RESUMEN

Algunos SNPs presentes en los genes µ-calpaína (CAPN1), calpastatina (CAST) y leptina (LEP) han sido asociados a la terneza de la carne de bovino, y con el fin de optimizar los procesos de mejoramiento genético en ganaderías de carne, es necesario adoptar metodologías rápidas, confiables y accesibles a los productores para determinar el genotipo de estos polimorfismos. Aunque existen varias metodologías, la HRM se plantea como alternativa promisoria que cumple con estas condiciones. Por lo anterior, se estandarizó y validó la PCR-HRM para la genotipificación de los SNPs CAPN4751, CAPN316, el CAST2959, CAST282, E2FB y E2JW en 50 animales de razas Bos taurus y Bos indicus, utilizando la PCR-RFLP, como prueba de oro. Se observó concordancia entre los resultados obtenidos por PCR-HRM, comparado con la PCR-RFLP, y se observó alta sensibilidad, especificidad y valores predictivos negativos y positivos adecuados para la PCR-HRM. De igual manera se corroboró la repetitividad y reproducibilidad al obtener homogeneidad en los resultados intracorrida e intercorrida. Aunque los resultados muestran que la PCR-HRM es una técnica eficiente y permite obtener resultados confiables en poco tiempo, sin embargo, éstos pueden ser afectados por el método de extracción, la calidad y cantidad del DNA, entre otros factores no determinados que pueden generar perfiles de disociación atípicos y falsos positivos. Por lo anterior se deben controlar estas variables y la reacción de PCR para identificar las curvas de disociación inusuales ocasionadas por exceso o deficiencia en la amplificación.

Palabras clave: Calidad, calpastatina, leptina, µ-calpaína, HRM.

INTRODUCTION

High resolution analysis of the dissociation temperature (HRM) of DNA fragments, amplified by the polymerase chain reaction (PCR), allows detecting single nucleotide polymorphisms (SNPs) (Druml and Cichna-Markl 2014), very fast and at a low cost, with high sensitivity, specificity and low contamination risking (Reed and Wittwer 2004 and Kyseľová et al. 2012). PRC-HRM is possible because of the development of sensors for detecting second generation dyes, which fluoresce when they are bound to double stranded DNA (dsDNA) and used in high concentrations to ensure saturation of dsDNA without inhibiting PCR (Gudnason et al., 2007). Diagnosis carried out by analyzing the melting temperature (Tm) and the profile of HRM, plotting the dissociation kinetics of dsDNA to single stranded DNA (ssDNA), when the temperature increases (Kristensen and Dobrovic 2008).

Therefore, taking into account the need of methodologies to improve productivity of animal husbandry sector in Colombia, the PCR-HRM was standardized and validated for determining the genotypes of SNPs related to tenderness of beef (CAPN316, CAPN4751, CAST282, CAST2959, LEP E2FB and LEP E2FW), using PCR-RFLP as gold standard test.

MATERIALS AND METHODS

Supported by the Comité Institucional para el Uso y Cuidado de los Animales (CICUA) from the CES University, the total DNA of peripheral blood was obtained from 25 Bos taurus and 25 Bos indicus animals.

In order to amplify by PCR, primers were designed, which surround fragments from 58 to 144 pb that potray the CAPN4751, CAPN316, CAST282, E2FB, and E2JW SNP (table 1).

Each SNP was analyzed independently, in a reaction mixture of 25 µL, containing 2 ng/µL of total DNA, 10 µM of primers, 12.5 µL of Master Mix HRM Genotyping PCR (QIAGEN, Hilden, Germany), diluted in water free of RNases. The amplification was performed in a Rotor-Gene 6000 thermal cycler (Corbett Life Science, Concorde, NSW, Australia), with preheating at 95 °C for 5 minutes, followed by 40 cycles, with a denatoration step at 95 °C for 10 seconds, a an annealing step, specific for each pair of primers for 30 seconds and one of elongation at 72°C for 10 seconds. The high resolution melt (HRM) profiles were built with the use of Rotor-Gene 6000 series© program (Corbett Life Science, Concorde, NSW, Australia), putting into a graph the fluorescence values, after exposing the amplified ones to a gradient with temperatures between 60 and 80 °C, which increased at a rate of 0.05 °C/s.

Sensitivity, specificity, predictive value of positive test, predictive value of negative test, repeatability, reproducibility and strength of genotype discrimination with HRM, were determined using RFLP as gold test, for each analyzed SNP.

RESULTS AND DISCUSSION

The efficiency of PCR-HRM was verified after comparing it with PCR-RFLP, and the SNPs genotype was determined (CAPN4751, CAPN316, el CAST2959, CAST282, E2FB and E2JW) in 50 Bos taurus and Bos indicus animals.

All genotyped samples, with PCR-RFLP, showed bands with the expected size in the amplification, after digestion of bovine genome fragments that carried CC genotypes of CAST282 and TT of E2FW.

Using PCR-HRM the differences between heterozygote and homozygote genotypes were demonstrated using the melting profile, evidenced in the normalized fluorescence curve, regarding the temperature (figure 1 A, C, G and I). On the other hand, homozygote genotypes were differentiated using a graph with normalized fluorescence, regarding temperature, in which a genotype was used as normalizer (figure 1. B, D, F, H, J, L) and based on melting temperature (Tm).

The SNP can be classified according to the type of homoduplex (A/T and G/C) or heteroduplex (A/G, A/C, T/C and T/G), which are produced after the amplification of a heterozygote (Liew 2004). Group 1 includes C/T and A/G transitions, with CAPN4751 and E2FB, presented by C/T, and CAST2959 with G/A. Group 2 shows C/A and G/T transversions and group 3 is C/G transversion, which includes CAPN316 and CAST282. Finally, group 4 is T/A transversion that classifies E2FW. SNP from group three and four were the most complex to differentiate, theoretically, there would be similar Tm values for heterozygote and homozygote due to the same pair of bases (C/G or T/A). Nevertheless, the HRM allows to differentiate them efficiently, as it was verified in this study.

There was an agreement among the results obtained by PCR-HRM, compared to PCR-RFLP. This previous fact demonstrates high sensitivity, specificity and proper positive and negative predictive values for PCR-HRM. Likewise, repeatability and reproducibility were demonstrated after obtaining homogeneity in the intra-run and inter-run results. The best results were reached with the use of HRM and amplification reaction conditions, described in the methodology. However, after evaluating the strength of the test, effect of the analyst skill was evident because variables like amount of DNA and reaction volume may affect the results.

The previous information confirms the efficiency of PCR-HRM as a promising molecular tool for determining the genotype of SNPs because of its high sensitivity, specificity and strength, similar to the suggestions of other researchers (Krypuy et al. 2006, Davoli et al. 2012 and Druml and Cichna-Markl 2014).

CONCLUSIONS

The potential of PCR-HRM was confirmed for determining genotype of SNPs CAPN4751, CAPN316, el CAST2959, CAST282, E2FB and E2JW, determining the genotype on 50 Bos taurus and Bos indicus animals. In all the samples evaluated by this technique, there was confirmation of the genotypes observed when using PCR-RFLP and sequencing, and 100% of sensitivity and specificity besides absence of false negative and false positive.

Results demonstrate that PCR-HRM is an efficient technique that allows to obtain reliable results in short time. However, it may be affected by the extraction method, DNA amount and quality, among other undetermined factors that may generate atypical melting profiles and false positives (unpublished data). Therefore, these variables should be controlled and PCR reaction should be monitored in order to identify unusual dissociation curves, provoked by excess or deficiency of amplification.

ACKNOWLEDGEMENTS

The authors thank to Dirección de Gestión del Conocimiento de la Universidad CES and to Colciencias (122870048969) for financing the research project that supported the results presented in this study.

REFERENCES

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Received: November 26, 2015 ]]> Accepted: April 7, 2016

L. E. López Rojas, Grupo Biología CES-EIA, Facultad de Ciencias y Biotecnología, Universidad CES, Colombia. Calle 10 A No. 22-4, Medellín, Colombia. Email: lelopez@ces.edu.co