Polymerase chain reaction amplification of nucleic acids was an enormous advance in the study of diseases and physiological cellular processes. The method was typically separated into 2 sequential phases of amplification by use of automated equipment and detection of the final product, or amplicon. Usually, detection involved the use of agarose or polyacrylamide electrophoresis gels for amplicon separation by size and visualization with fluorescent dyes.1,2 Currently, amplicon amplification and detection can occur simultaneously thanks to specific dsDNA intercalating dyes such as ethidium bromide, SYBR Green I, LC Green, SYBR Gold, PicoGreen, TOTO-1, Syto9, EvaGreen, and YOYO-1.3,4 The addition of one of those dyes to the PCR reaction results in the generation of fluorescence proportional to the amount of dsDNA present in the reaction tube. However, fluorescence is also produced by undesirable reactions such as primer dimerization. Because the dye-associated fluorescence is emitted only when the dye is intercalated to dsDNA, the denaturation of the double strands causes a reduction in the fluorescence signal. A plot of the fluorescence signal by temperature generates a melting curve for the amplicon, which is dependent on its GC content, length, and sequence.2
High-resolution melting analysis is a closed-tube, fluorescence-based, genotyping and mutation scanning method that was developed by the University of Utah and Idaho Technology in 2002.5 It is a simple, rapid, and inexpensive technique that is dependent on PCR design and the specific DNA intercalating dye and instrument used for detection of fluorescence. The procedure consists of a conventional PCR assay followed by a melting step. High-resolution melting analysis is an excellent technique for detection of SNPs, but it is not a good technique for detection of insertions and deletions.4,6
Myostatin, also known as growth differentiation factor 8, is a peptide hormone that is responsible for negative regulation of skeletal muscle growth.7 It is present in all skeletal muscle and targets satellite cells, the mononuclear progenitors of muscle lineage within muscle fibers, keeping them in a quiescent state.8 Naturally occurring mutations in the MSTN have been identified in horses,9,10 dogs,11 sheep,12,13 cattle,14–18 mice,19,20 and humans.21 Those mutations can decrease or interrupt myostatin production, and individuals with a mutation have a greater muscle mass, compared with individuals without a mutation. Mutations in the MSTN also affect growth, reproduction, performance, and carcass quality.9 In Thoroughbreds, a novel SNP (g.66493737C>T) in the MSTN is highly correlated with the optimal race distance for individual horses.10 For example, horses with the C/C genotype of MSTN perform best in fast short-distance races, whereas horses with the T/C genotype compete best in middle-distance races, and horses with the T/T genotype have the most stamina and perform best in long-distance or endurance races.10 The frequency of each of those 3 genotypes varies among breeds of horses; 28 of 31 (90%) Egyptian Arabians (horses known for their endurance) evaluated had the T/T genotype, whereas 29 of 35 (83%) Quarter Horses (horses used for short-distance racing and activities requiring short bursts of speed) evaluated had the C/C genotype.10 Other studies22,23 have been conducted to investigate the frequency of the C allele among different breeds of horses that have been bred specifically for racing, trotting, endurance, or draft purposes as well as ponies and other equine breeds that have been selected for resistance to certain climatic conditions; however, to our knowledge, no studies have been conducted on horses that perform in various athletic competitions that require jumping.
The SNP in the MSTN described by Hill et al10 can be used as a molecular marker to predict athletic performance of horses and may be used in the future as a tool for genetic selection of horses for specific sports. The objective of the study reported here was to develop an HRM assay to detect the g.66493737C>T polymorphism in the MSTN and determine the frequency of each of the 3 previously defined MSTN genotypes (T/T, T/C, and C/C) in a population of warmblood horses. We hypothesized that the HRM analysis developed would be sufficiently accurate for detection of the g.66493737C>T polymorphism such that it could be used in lieu of standard PCR genotyping methods and that the frequency of the C/C genotype in warmblood horses would be low, whereas the frequencies of the T/T and T/C genotypes would be similar.
Materials and Methods
Animals and sample collection
All study protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Federal University of Rio Grande do Sul and adhered to the principles for the humane treatment of animals. The study population consisted of 23 clinically normal warmblood horses of various breeds (Brazilian Sport Horse, n = 17; Belgian Warmblood, 3; and Hanoverian, 3) that competed in athletic activity that required jumping (ie, jumpers). The population included 13 males and 10 females with ages that ranged between 4 and 17 years. All horses were owned by private individuals, and consent was obtained from all horse owners prior to collection of study samples.
From each horse, a venous blood sample (3 mL) was collected by jugular venipuncture directly into sterile evacuated blood collection tubes that contained EDTA as an anticoagulant. Genomic DNA was extracted and purified from each sample by use of commercial kitsa with silica columns. The amount of DNA extracted from each sample was measured by fluorometry. Samples of DNA for a Quarter Horse with the C/C genotype, Arabian with the T/T genotype, and Thoroughbred with the C/T genotype obtained from the DNA database of the Laboratory of Animal Pharmacogenetics (Laboratório de Farmacogenética Animal, UFRGS, Porto Alegre, RS, Brazil) were used as positive control samples.
Polymerase chain reaction assay and direct sequencing of MSTN
To sequence the g.66493737 portion of the MSTN, primers were designed by use of a commercially available primer design tool.b The goal during primer design was to generate a product with < 150 bp for analysis with HRM. The resulting primersc (forward primer, 5′-GACACAACAGTTTCAAAATATTGTTCTCCTT-3′; and reverse primer, 5′-CCAGGACTATTTGATAGCAGAGTCA-3′) yielded a 98-bp amplicon.
The PCR reaction mixture for sequencing of the g.66493737 portion of the MSTN included approximately 20 ng of sample DNA, 1X PCR buffer,d magnesium chloride solution (concentrations ranging from 0.75 to 3mM), 0.5 U of Taq polymerase, 0.1mM of each dideoxynucleotide, 10 pmol of each primer, and water in a volume sufficient to bring the volume of the mixture to 25 μL. The assay was conducted with a thermocyclere and consisted of initial denaturation at 94°C for 3 minutes; 35 amplification cycles of 94°C for 30 seconds; annealing at temperatures of 55°, 57°, or 59°C for 30 seconds; extension at 72°C for 1 minute; and a final extension cycle at 72°C for 10 minutes. An aliquot of the resulting amplification product was visualized on a native polyacrylamide 12% gel after electrophoresis at 100 V for 60 minutes and silver staining.24 The amount of DNA present after PCR was measured by fluorometry.
The PCR products were purifiedf before sequencing. Samples were sequenced at the Centro de Pesquisa Experimental, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil, by use of a capillary automated sequencer.g
HRM analysis
The HRM analysis was performed with a commercially available kith in accordance with the manufacturer's instructions. Briefly, 3 different protocols were tested to assess which one had the best reproducibility. The reaction mixture for the first protocol consisted of 20 ng of sample DNA, 1X PCR master mix, 0.7μM of each primer, and water in a volume sufficient to bring the volume of the mixture to 25 μL. For the second protocol, 1 μL of a solution with a high-salt concentration (1M potassium chloride and 0.5M Tris-hydrochloride; pH, 8) was added to the reaction mixture before the first melting cycle. For the third protocol, an unlabeled probe with the 3′ end blocked by a 3-carbon spacer (5′-CAGGTTATAATG CACCAAATAATTTTC/3SpC3/-3′) was added to the reaction mixture such that the mixture contained 5 parts reverse primer, 4 parts probe, and 1 part forward primer. All samples were assessed in triplicate for each protocol, and each protocol was analyzed once. The analysis was conducted with a thermocycleri and consisted of an initial enzyme activation step at 95°C for 5 minutes, followed by 40 cycles of denaturation at 95°C for 10 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 10 seconds. Immediately after the amplification step, the HRM thermocycler was programmed to increase the temperature from 65° to 95°C in 0.1°C-increments every 2 seconds for data acquisition and determination of the Tm.
Statistical analysis
A software program associated with the HRM thermocycleri was used to calculate the R2 and provide the level of confidence for each curve relative to the positive control curves. The fluorescence threshold was determined manually. The linear fluorescence intensity before and after the melting transition of each sample was defined (baseline) so the fluorescence intensity values during the melting transition could be normalized between 0% and 100%.
Results
The optimal annealing temperature and magnesium chloride concentration for the PCR assay were 55°C and 1.5mM, respectively. Use of the primers designed to detect the g.66493737 portion of the MSTN for this study resulted in DNA sequencing for 20 of the 23 horses evaluated; the chromatograms were uninterpretable for the remaining 3 horses. A g.66493737C>T polymorphism was not detected in any of the 20 sequenced samples.
Use of the designed primers alone or in combination with a high-salt solution during HRM analysis did not result in consistent melting curves. Reaction mixtures that contained an unlabeled probe with the 3′ end blocked by a 3-carbon spacer (third protocol) did result in consistent melting curves (ie, the shape of the curve was highly reproducible among the 3 replicates for each sample). Consequently, the HRM analysis results presented in this report were derived by use of the third protocol.
The MSTN genotype was determined by HRM analysis for 16 of the 23 horses evaluated. The genotype determined by the HRM analysis was in agreement with that determined by standard PCR methods for 14 of those 16 horses (Supplemental Table S1, available at http://avmajournals.avma.org/doi/suppl/10.2460/ajvr.78.1.63). The raw data generated by the HRM analysis was normalized for correction of nonspecific signal and plotted against temperature (Figure 1). The first 10 cycles for each sample were eliminated from the analysis, and the threshold was set at 0.04192. For the remaining 7 horses, the MSTN genotype could not be determined by HRM analysis when the cutoff level of confidence relative to the respective positive control curves was set at 70%. Of those 7 horses, the MSTN genotype determined by standard PCR methods was T/T for 3, T/C for 3, and undetermined for 1. The differences in the normalized fluorescence intensity between an individual sample and the normalized fluorescence intensity of the positive control for the T/T genotype throughout the melting transition of the HRM analysis were summarized (Figure 2).
For the 11 horses with the T/T genotype as determined by HRM analysis, the mean threshold cycle for the 3 samples analyzed for each individual ranged from 23.9 to 26.15, and the mean level of confidence of the HRM curve relative to the HRM curve for the positive control for that genotype ranged from 74.0% to 96.8%. For the 4 horses with the T/C genotype, the mean threshold cycle for the 3 samples analyzed for each individual ranged from 22.99 to 25.29 and the mean level of confidence of the HRM curve relative to the HRM curve for the positive control for that genotype ranged from 79.2% to 88.5%. For the horse with the C/C genotype, the mean threshold cycle was 24.5 and the mean level of confidence of the HRM curve relative to the HRM curve for the positive control for that genotype was 96.7%.
Of the 22 horses for which MSTN genotype was determined by standard PCR methods or HRM analysis, 14 (63.6%) were homozygous T/T, 7 (31.8%) were heterozygous T/C, and 1 (4.5%) was homozygous C/C. The frequency of alleles T and C in the study population was 79.5% and 20.5%, respectively.
Discussion
To our knowledge, the present study was the first to determine the prevalence of each of the 3 MSTN genotypes by use of HRM analysis in a population of warmblood horses. The HRM technique developed in this study was able to determine the MSTN genotype for 16 of the 23 (70%) horses evaluated with a mean confidence level of 84.4%. The study population consisted of warmblood horses that competed in activities that required jumping (jumpers). Only 1 horse in this study had the C/C genotype, which is most commonly associated with horses that excel at short, fast racing. This was an expected finding because the physical conformation required for jumping is not particularly compatible with that required for short bursts of speed. We hypothesized that the proportion of the study population with the T/C genotype would be similar to that with the T/T genotype because both genotypes are associated with desirable characteristics for jumpers such as weightlessness and power for propulsion. However, of the 22 horses for which the MSTN genotype was determined, only 7 (31.8%) had the T/C genotype, whereas 14 (63.6%) had the T/T genotype. The proportion of horses with the T/T genotype in the present study was less than that reported for Egyptian Arabians in another study.10
The primers used for the PCR and HRM analyses in the present study were designed to yield an amplicon as small as possible (98 bp) because the sensitivity and specificity of the HRM analysis are optimized for amplicons with ≤ 300 bp. Moreover, long amplicons melt in 2 stages and generate complex melting curves.25
Use of the designed primers alone or in combination with a high-salt solution during HRM analysis did not result in consistent melting curves. All samples were processed in the same manner; therefore, common sources of variation during HRM analysis such as inadequate PCR product length, ionic strength of the buffer solution, variability in the DNA extraction method, and instrument quality of data acquisition5,26 were controlled and should not have contributed to the generation of inconsistent curves. The addition of a buffer solution with a high salt concentration to the reaction mixture before a second melting phase can sharpen and increase the resolution and clustering of the curves generated during HRM analysis, albeit in a somewhat unpredictable manner.26 However, that did not happen in the present study.
Consistent and reproducible melting curves were only generated when an unlabeled probe with the 3′ end blocked by a 3-carbon spacer was added to the reaction mixture such that the mixture contained 5 parts reverse primer, 4 parts probe, and 1 part forward primer. The use of sequence-specific probes with labeled substances such as fluorescein amidite and hexochloro-fluorescein is highly efficient but more costly than the use of unlabeled oligonucleotides.27,28 The 3′ end of an unlabeled probe is blocked to prevent polymerase extension, which yields 2 amplicons (1 amplicon with the appropriate [designed] size and a smaller amplicon)/analyzed sample and an extra asymmetric curve to help to differentiate the genotypes. Blocking of the 3′ end of an unlabeled probe can be accomplished with 3′-phosphorylation, 2′3′-dideoxynucleotide, 3′-deoxynucleotide, 3′-3′ linkage, inverted dT, amino-modifier C6, 3-carbon spacer, or mismatching the last two 3′ bases. We chose to use a 3-carbon spacer because of its reported stability.27,29,30 Instead of generating an additional visible curve in the temperature range of data acquisition, the unlabeled probe aided in the generation of curves that could be distinguished from each other on the basis of temperature, a finding that, to our knowledge, has not been previously reported. Ideally for HRM genotyping, the GC content of the target amplicon should be between 40% and 60%, the Tm for the probe should be approximately 10°C greater than the Tm for the primers, and the Tm for the probe and primers should be > 56°C.6,31–33 Those recommendations were not met in the present study, most likely because the GC content of the target amplicon was < 40%.
The inconsistency among the melting curves for the 7 horses for which the MSTN genotype could not be determined by HRM analysis might have been caused by polymorphisms within the amplicon, which altered the Tm. However, sequencing results of the g.66493737 portion of the MSTN as determined by standard PCR methods did not reveal any polymorphisms, and the cause of the aberrant melting behavior during HRM analysis for the samples obtained from those horses could not be determined. Two other pairs of primers (one pair of primers yielded a 158-bp amplicon, and the other pair yielded a 108-bp amplicon) were used for HRM analysis of the positive control samples used in the present study in an attempt to determine the MSTN genotype, but the resulting curves had poor reproducibility (data not shown). Therefore, we concluded that the primers that yielded the 98-bp and the unlabeled probe used in this study were the best alternative for HRM analysis of the MSTN genotype. The high adeninethymine content within the g.66493737 region of the MSTN makes the design of primers and probes suitable for HRM analysis challenging. Also, unlabeled probes can have incomplete blocking of the 3′ end, which will contribute to the generation of aberrant melting curves. Although the 3-carbon spacer used in the present study has one of the best blocking efficiencies, it is not 100% effective. The genotype could not be determined by sequencing or HRM analysis for 1 horse in the present study. That horse may have had a substance in its blood that inhibited DNA extraction.34
In the present study, HRM analysis was able to successfully determine the MSTN genotype for 16 of the 23 (70%) warmblood horses evaluated, and the genotype determined by HRM analysis was in agreement with that determined by sequencing for 14 of those 16 horses. Given that the cost of sequencing is approximately 3 times that of HRM analysis,35 results of the present study suggested that HRM analysis might be a viable alternative for use to further investigate the g.66493737C>T polymorphism of the MSTN and the performance of jumpers. Determination of the MSTN genotype of a young horse may be useful as a predictor of the type of athletic activity for which it is best suited before training is initiated. High-resolution melting analysis is a closed-tube technique, which decreases the operational risks associated with the manipulation of biohazardous materials such as ethidium bromide and polyacrylamide. The use of alternative primers to yield a differently sized amplicon and an unlabeled probe with a higher Tm than those used in the present study might improve the efficacy of the HRM technique for identification of the g.66493737C>T polymorphism and the MSTN genotype.
Acknowledgments
This manuscript represents a portion of a thesis submitted by Dr. Serpa to the Programa de Pós-Graduação em Medicina Animal: Equinos (PPGMAE), Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul (UFRGS), as partial fulfillment of the requirements for a Doctor of Science degree.
Supported by the National Council for the Improvement of Higher Education (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES).
The authors declare that there were no conflicts of interest.
Drs. Serpa, Garbade, and Natalini contributed to study design, study execution, data analysis and interpretation, manuscript preparation, and final approval of the manuscript. Drs. Pires and Tisotti contributed to study execution and final approval of the manuscript.
ABBREVIATIONS
dsDNA | Double-stranded DNA |
GC | Guanine-cytosine |
HRM | High-resolution melting |
MSTN | Myostatin gene |
SNP | Single-nucleotide polymorphism |
Tm | Melting temperature |
Footnotes
PureLink Genomic DNA Mini Kit, Invitrogen, Carlsbad, Calif.
EquCab, version 2.0, National Center for Biotechnology, Bethesda, Md. Available at: www.ncbi.nlm.nih.gov. Accessed Jan 14, 2014.
Integrated DNA Technologies Inc, Coralville, Iowa.
Recombinant Taq DNA Polymerase, Invitrogen, Carlsbad, Calif.
Veriti 96-Well Thermal Cycler, Applied Biosystems, Foster City, Calif.
Wizard SV Gel and PCR Clean-Up System, Promega, Madison, Wis.
ABI 3500 Genetic Analyzer, Applied Biosystems, Foster City, Calif.
Type-it HRM PCR Kit, Qiagen, Hilden, Germany.
Rotor-Gene Q, Qiagen, Hilden, Germany.
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