Abstract
Los análisis genómicos y transcriptómicos para selección y mejoramiento genético animal requieren ADN o ARN de alta concentración y pureza, proveniente de diferentes tejidos incluyendo semen. Los métodos usualmente utilizados para extraer ADN de semen son menos efectivos en cantidad y calidad de ADN, debido a los solventes y diluyente utilizados para la conservación, características físico-químicas de los espermatozoides, y fracción no celular del eyaculado. En este estudio, se proponen modificaciones al método de tiocianato de guanidina, incluyendo un segundo lavado de la muestra con solución buffer fosfato, y dos lavados con solventes orgánicos, uno fuerte (fenol:cloroformo:alcohol isoamílico) y uno débil (cloroformo:alcohol isoamílico), para retirar la proteína y diluyente presentes en la muestra. Además, se propone una incubación por separado con ARNasa para reducir contaminación de ácidos nucleicos en la medición y elaboración de diluciones para amplificación por PCR. La precipitación agregó al isopropanol de la metodología original 3 M de acetato de sodio para retirar restos de posibles inhibidores de la PCR. Finalmente, se incluyeron centrifugaciones de alta velocidad y decantaciones para evitar la necesidad de separación mecánica del ADN y la proteína. El ADN extraído con el método propuesto no presentó degradación, y la calidad y cantidad fueron mejores (P<0.0001), encontrándose una media de 1.84±0.09 en el rango 260/280 y 156.99±7.29 ng/ꟺ para la variable de concentración. El presente método de extracción es una alternativa de bajo costo, viable para obtener ADN de semen con características necesarias para análisis genómicos en mamíferos.
Highlights
Genetic analysis is widely used in domestic animals for the identification and comparison of genetic variations within a population
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 sperm(1,2,3)
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
Summary
Genetic analysis is widely used in domestic animals for the identification and comparison of genetic variations within a population. 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. 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). 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 PCR(7,8,9). 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
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