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Revista mexicana de ciencias pecuarias

versión On-line ISSN 2448-6698versión impresa ISSN 2007-1124

Rev. mex. de cienc. pecuarias vol.7 no.4 Mérida oct./dic. 2016

 

Articles

Modification of a guanidinium thiocyanate to extract DNA from semen for genomic analysis in mammals

Baldomero Alarcón-Zúñigaa 

José Luis Zepeda-Batistaa 

Agustín Ruíz-Floresa  * 

Luis Joaquín Gómez-Mezaa 

José Guadalupe García-Muñiza 

Rafael Núñez-Domíngueza 

Rodolfo Ramírez-Valverdea 

Itzel Villegas-Velázqueza 

a Posgrado en Producción Animal, Departamento de Zootecnia, Universidad Autónoma Chapingo, Carretera México-Texcoco km 38.5. CP 56230, Chapingo, Estado de México. México.

Abstract:

The genomic and transcriptomic analyses for selection and genetic improvement require DNA or RNA of high concentration and purity, coming from different tissues, including semen. The DNA may be from hair, saliva, cartilage, blood, or semen. The methods usually used to extract DNA from semen have low efficiency in terms of quantity and quality. This is due to the solvents and dilutors used for semen conservation, physical and chemical characteristics of sperms, and non-cellular fraction of ejaculate. In this study, modifications to the guanidinium thiocyanate method are proposed, including a second washing of the sample using a phosphate buffer solution, and two washing with organic solvents, one strong (phenol:chloroform:Isoamyl alcohol), and one weak (chloroform:isoamyl alcohol), to get rid of the protein and dilutors present in the sample. Additionally, it is proposed a separate incubation with RNA-ase to reduce contamination of nucleic acids during measurement and dilution preparation for PCR amplification. The precipitation added to the isopropanol of the original method 3 M sodium acetate to separate the residuals of potential PCR inhibitors. Finally, there were included high-speed centrifugation and decantation to avoid the need for mechanical separation of the DNA and protein. The DNA extracted with the modified method did not present degradation, and the quality and quantity were better (P<0.0001) than the original, with a mean of 1.84±0.09 in the range of 260/ 280 and 156.99±7.29 ng/µl for concentration. The present method of extraction is a viable low-cost alternative to obtain DNA from semen with the necessary characteristics for genomic analysis in mammals.

Key words: Genomic DNA; DNA extraction; Semen; Mammals

Introduction

Genetic analysis is widely used in domestic animals for the identification and comparison of genetic variations within a population. It is a vital component of techniques such as DNA sequencing, structural variation, genotyping, gene expression and identifying regulatory or functional elements. Techniques for genetic analysis typically require large amounts of highly pure DNA. Genetic source material (e.g. follicle tissue, saliva, blood, cartilage, semen) determines the methods most appropriate for genetic analysis1.

Unlike DNA extraction from somatic cells, with semen there are a number of difficulties in the purification methodology; these involve the physical and chemical characteristics of spermatozoid nuclear compaction, composition of the ejaculate's non-cellular fraction, and the diluents used in the preservation of frozen sperm1,2,3. Spermatozoids contain specialized, low-molecular weight nuclear proteins called protamines. These generate a chromatin that is at least six times denser than the histones of somatic cells and that maintain the DNA cells condensed in the acrosome. In addition, their plasmatic membrane is linked by disulfide bonds, making it resistant to the chemical agents used in trADNtional somatic cell DNA extraction methods4,5. The spermatozoids' acrosome contains hyaluronidase, an enzyme that attacks hyaluronic acid when it contacts the ovule. In DNA extraction, however, hyaluronidase is rel eased wh en th e pl asm atic membrane ruptures, possibly degrADNng the DNA. Another possible way DNA can be damaged is when spermatozoid connector mitochondria are lysed and released because they preserve their oxidative activity and can damage any DNA in the medium6,7.

The non-cellular fraction of the ejaculate contains a number of minerals, such as zinc and copper, which originate in the prostate. If these are not removed from the final DNA sample, they interfere in the polymerase chain reaction (PCR). Moreover, glycogen and some lipids in the ejaculate can function as energy sources6,7. Finally, when semen is frozen, the diluents added to preserve it simulate the composition of the non-cellular fraction. They contain proteins, lipids and minerals (e.g. Cr, Fe, Zn, Cu, Cl, K, P, Ca, Mg, Na, and S), making it necessary to remove them completely from samples before DNA extraction to prevent any interference with the PCR7,8,9.

The present proposed technique is based on the guanidinium thiocyanate method, and other techniques for extracting DNA from frozen semen, which result in DNA samples of the necessary quality and quantity for current genetic identification techniques. It completely eliminates the need to mechanically separate the remaining protein; a required step in existing techniques because this protein can lead to widely varying reADNngs between samples if not removed7,10,11. The present study objective was to develop and evaluate a technique based on the guanidinium thiocyanate method that can efficiently extract high-molecular weight genomic DNA from frozen bull semen for use in genetic identification techniques that require high purity and operate within values of 1.8 to 2.0 in the 260/280 wavelength range, and 2.0 to 2.2 in the 260/230 range.

Material and methods

All analyses were performed in the Laboratorio de Genética Molecular of the Departamento de Zootecnia, Universidad Autónoma Chapingo.

Biological material

DNA extraction was done using 0.25 ml straws of commercial frozen bovine semen, and 0.5 ml straws of frozen semen prepared in the laboratory. The lab-prepared straws were tested with a commercial diluent (Triladyl®; contains TRIS, citric acid, sugar, buffers, glycerin, ultrapure water, antibiotics and egg yolk). Extraction tests were done using frozen semen straws from five bulls (1 Holstein, 4 Jersey).

DNA extraction

Extraction was done using three methods: a) phenol -ch l oroform 12; b) guanidinium thiocyanate; and c) the combination method proposed in the present study. Each technique was run using both the commercial and lab-prepared straws, which were prepared in the same manner. After thawing, the semen was washed with 1 ml saline phosphate buffer solution, centrifuged at 800 rpm for 10 min at room temperature, and then washed again in the same way. The resulting pellet was incubated with 1 ml lysis solution (6 M guanidinium thiocyanate, 30 mM sodium citrate [pH 7.0], 0.5% sarkosyl, 0.20 mg/ml proteinase K and 0.3 M ß-mercaptoethanol) for 4 h at 55 °C in a water bath. When incubation was complete, the supernatant was recovered, washed with 1 ml FCl solution (25, phenol: 24, chloroform: 1, isoamyl alcohol), and centrifuged at 9,000 rpm for 10 min at 4 °C. This procedure was repeated with the Cl solution (24, chloroform: 1, isoamyl alcohol) and the supernatant incubated with RNAse for 30 min at 37 °C. It was then precipitated with cold isopropanol and 0.1 volumes of 3 M sodium acetate, and incubated overnight at -20 °C. It was then centrifuged at 9,000 rpm for 10 min at 4 °C, the supernatant decanted and the pellet washed with cold 70% ethanol. Finally, it was centrifuged, the supernatant decanted, the remaining contents allowed to dry, and these resuspended in 50 μl TE buffer.

DNA analysis

For all three methods, DNA concentration was evaluated with a nano-spectrophotometer (ND-1000 Nanodrop®), using the 260/230 and 260/280 wavelength ranges. Quality was determined by electrophoresis in 1% gel to measure sample contamination and degradation.

Statistical analysis

For all 65 samples, DNA concentration and quality in each method were initially analyzed using the effects of breed and individual, and the covariable of semen straw volume. None had a significant effect and were therefore eliminated from the model. The final analysis considered only the effect of extraction method (1= phenohchloroform; 2= guanidinium thi ocyan ate; 3= modified guanidinium thiocyanate). All analyses were run with the GLM procedure in the SAS statistical package13.

Results and discussion

The least means squares analysis identified differences (P<0.0001) between methods in the 260/280 wavelength range and between sample concentrations (Table 1). This confirms that the modified method extracted a higher quality and quantity of DNA compared to the methods of Hossain et al10 and Birren et al12. Absence of difference in the 260/230 range indicates total cleansing of RNA from the samples was attained in all three extraction methods. These results are supported by the electrophoresis analysis results of the DNA samples extracted with each evaluated method (Figure 1).

Table 1 Least means squares (± standard error) results for quality and quantity of DNA extracted from frozen bull semen using three methods 

† Birren et al, 1997; ¶ Hossain et al, 1997; § Developed in present study.

abc Least squares means with different letters in the same column are different (P<0.05).

Phenol-chloroform (lanes 1 to 4); guanidinium thiocyanate (lanes 5 to 8); and modified guanidinium thiocyanate (lanes 9 to 12). "M" lanes are 1 kb molecular weight markers.

Figure 1 1% gel electrophoresis of bovine semen prepared with one of three DNA extraction methods 

When extracted with the phenol-chloroform method, DNA samples exhibited low quality (1.47 ± 0.11 at 260/280; and -0.41 ± 0.41 at 260/230), and low concentrations. Electrophoresis demonstrated an absence of DNA, possibly due to degradation and loss from total washing during the protocol. This coincides with previous studies1,10 in which DNA degradation was extensive or complete when usi ng the phenol-chloroform method12. However, in these same studies DNA quality surpassed 1.8 at 260/280, possibly due to the presence of RNA and the remains of low-molecular weight proteins.

The guanidinium-thiocyanate method produced good quality DNA samples with higher concentrations than in the phenol-chloroform method. Nonetheless, electrophoresis showed slight DNA degradation in the samples and considerable presence of protein in the gels' upper fraction (Figure 1, rows 5 to 8). Slight DNA degradation and the presence of protein were also observed in samples in a previous study10, partially due to the need for DNA separati on by protei n tracti on , causin g incomplete purification of the nucleic acids.

The modified guanidinium-thiocyanate method proposed here produced high quality and quantity DNA samples. Nano-spectrophotometry showed quality to be between 1.8 and 2.2 in both wavelength ranges (i.e. 260/230 and 260/ 280) (Table 1). Little or no DNA degradation was apparent in the electrophoresis analysis, with a total absence of proteins and other contaminating factors (Figure 1, rows 9 to 12). The samples provided optimum conditions for use in genetic analysis.

Extraction efficiency from frozen bovine semen samples using the modified method proposed here was compared to the efficiency reported in previous studies10,12. In the proposed method, a second washing with phosphate buffer solution was added to the previous methods to increase DNA purity. This was done even though livestock species normally produce more ejaculate than humans and its density is 0.2 to 30.0 times greater6. In addition, bull, pig and horse ejaculates have Cl, K and Ca concentrations 1.7 to 3.4 times higher than human ejaculate. If these ions are not removed from semen samples they can cause erroneous results during genetic analyses and even inhibit PCR amplification of DNA7,9.

In the proposed method, two washings with organic solvents followed by high velocity centrifuging were used even though use of organic solvents is reported to make isolation of non-degraded DN A more difficult14. However, this modification helped to completely remove the protein that had not degraded in previous steps. It also aided in preserving DNA integrity by consolidating it into a pellet, thus preventing chemical damage by completely separating it from the organic solvent, any compounds in the ejaculate, and any diluents added for semen preservation. Addition of the phenol:chloroform:isoamyl alcohol washing step removed high-molecular weight proteins and residual chelating agents in the samples while the subsequent chloroform:isoamyl alcohol washing removed low-molecular weight proteins and any chelant traces remaining after the first washing7,9.

Since Hossain et al10 reported that RNA material was preserved in some samples, an RNAase incubation was added in the modified method. This ensured the DNA in the samples was genetically pure, thus avoiding imprecise values when measuring concentrations by nano-spectrophotometry. If values are not exact, DNA sample dilution calculations can contain errors which affect PCR amplification results7.

Finally, use of 3 M sodium acetate in DNA incubation with isopropanol at -20 °C made DNA precipitation more efficient by augmenting solution ionic strength and removing organic solvents and diluent medications8. Centrifuging, decanting and washing with 70% ethanol removed all sodium acetate and sarkosyl remnants.

Conclusions and implications

The modified DNA extraction method proposed here for use with frozen bovine semen produced highly pure DNA samples free of degradation and with extremely low or null protein content. It is also simple and low-cost, and the resulting sample DNA quality and quantity are appropriate for genetic analyses, such as SNPs and microsatellites, that are commonly used in mammals.

Acknowledgements

The research reported here was financed by the Consejo Nacional de Recursos Genéticos Pecuarios (CONARGEN) (2013-001), Asociación Mexicana de Criadores de Ganado Jersey de Registro and the Universidad Autónoma Chapingo, México (DGIP-11550301). The Consejo Nacional de Ciencia y Technologia (CONACYT) provided a Masters in Science scholarship for the second author.

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Received: June 11, 2015; Accepted: November 23, 2015

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