Protocol for extraction of genomic DNA from swine solid tissues

Protocol for extraction of genomic DNA from swine solid tissues

Fernando Henrique Biase, Maurício Machaim Franco, Luiz Ricardo Goulart and Robson Carlos Antunes

Universidade Federal de Uberlândia, Instituto de Genética e Bioquímica, Uberlândia, MG, Brazil

ABSTRACT

Molecular diagnostics are performed by using DNA from different body tissues. However, it is necessary to obtain genomic DNA of good quality. Due to the impossibility of collecting blood from slaughtered animals, DNA extraction from solid tissues is necessary. The objective of this study was to describe a protocol of DNA extraction from swine skin, adipose, brain, liver, kidney and muscle tissues. We obtained high molecular weight DNA of good quality, shown by agarose gel and amplification of two DNA fragments, 605bp and 891pb, by PCR. Spectrophotometric analysis of DNA concentration showed variation among the DNA from different tissues, with the liver and adipose tissues presenting the greatest and the smallest concentration, respectively. The described protocol has proven to be advantageous due to its simplicity, quickness, affordable reagents and absence of phenol, resulting in a high molecular weight DNA of good quality from several tissues.

Key words: DNA extraction, swine tissues.

Received: July 31, 2000; accepted: August 1, 2002.

INTRODUCTION

Professionals that control swine meat and carcass quality work basically with slaughtered animals, and it is not possible to collect blood for DNA extraction when genetic exams are needed. For this reason, DNA must be extracted from solid tissues.

Many genomic DNA extraction protocols have been described for prokaryotes and eukaryotes, from cell sample to specific tissues (Sambrook et al., 1989). However, there are not many studies using DNA from swine solid tissues.

Like reagents, good quality DNA is essential to achieve good results in experiments (Hoy, 1994), especially in the Polymerase Chain Reaction (PCR), in which excess of cell debris and proteins may inhibit the amplification process (Saiki, 1990).

Due to the importance of having good quality DNA extracted from solid tissues, this paper describes a fast and simple protocol for genomic DNA extraction from swine skin, adipose, brain, liver, kidney and muscle tissues.

MATERIAL AND METHODS

Three samples from skin, adipose, brain, liver, kidney and muscle tissues were stored at -20 °C after slaughter, from which DNA was extracted.

A sample consisted of a 200 mg biopsy. Specimens were ground separately with mortar and pestle in liquid nitrogen. The frozen powder was transferred to a 2 mL eppendorf tube and 800 mL of extraction solution (50 mM Tris-HCL, pH8.0; 25 mM EDTA and 400 mM NaCl), 100 mL 10% SDS, and 20 mL Proteinase K (10 mg/mL) were added. The extract was homogenized and incubated at 65 °C for 3 h. After incubation, proteins and cellular debris were precipitated by adding a 300 mL 6 M NaCl, kept at 4 °C for 15 min. Centrifugation was done at 25,000 g for 20 min. 500 mL of the supernatant were transferred to a new eppendorf, with 500 mL 8 M guanidine hydrochloride (pH 8.0), and 0.49 M ammonium acetate solution, and kept in mild agitation for 90 min. Nucleic acids were precipitated by adding 800 mL of cold 100% isopropyl alcohol, followed by centrifugation at 8,000 g for 5 min. Pellets were washed with 400 mL of 70% isopropyl alcohol. After drying, pellets were resuspended in 150 mL TE buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA and 50 mg/mL RNAse). DNA samples were stored at 4 °C.

DNA quality and concentrations were evaluated by spectrophotometer and by ethidium bromide-stained agarose gel under Ultraviolet light. Photographs were documented through a Videodocumantetion system, the VDS ImageMaster (Pharmacia Biotech).

Fragments chosen to probe DNA integrity are in different regions of the porcine Growth Hormone gene. A 605pb fragment was amplified under the following conditions: 100 ng of genomic DNA, 1U Taq DNA Polymerase, 0.5 mM Primer, 400 mM dNTP, Taq buffer 1X and 3 mM MgCl₂. Another fragment, 891bp, was amplified in the following conditions: 100 ng genomic DNA, 1U Taq DNA Polymerase, 0.2 mM Primer, 400mM dNTP, Taq buffer 1X and 1.5 mM MgCl₂. Both in a 20 mL final volume.

RESULTS AND DISCUSSION

The protocol was efficient in extracting genomic DNA from all solid tissues. Analysis of whole genomic DNA in agarose gel (Figure 1) and amplified fragments (605 and 891bp) by PCR (Figures 2a and ^b) demonstrated that the extracted DNA had high molecular weight, one of the most important aspects for successful amplifications of larger fragments (Borges, 1997).

Spectrophotometer measurements indicated differences in DNA concentration and purity, according to the tissue origin (Table I). Higher concentrations were obtained from the liver, kidney and brain tissues, similar to those obtained by Atmadja et al.(1995) for human tissues. The OD260/OD280 ratio values satisfied those suggested by Sambrook et al. (1989), ³ 1.8, which were higher than those obtained by Akane et al. (1993) and Atmadja et al.(1995).

The variability in DNA quality and purity can be explained by tissue specific structural complexity. Liver, kidney and brain tissues are composed of delicate membrane cells with few fibrous cells. On the other hand, the skin consists of stratified tissue with keratin and other fibrous cells. The muscle tissue is constituted by many proteins within the cell. Adipose tissue consists mainly of lipids, which increase cell volume and decrease cell number. In addition, lipid is water insoluble, which hinders extraction, lysing a smaller number of nuclei (Junqueira and Carneiro, 1995).

This protocol proved to be advantageous because of its simplicity, quickness and affordable reagents, besides the high molecular weight DNA and purity achieved in a variety of tissues. Furthermore, there is no phenol in DNA purification, known as a strong PCR inhibitor (Saiki, 1990).

ACKNOWLEDGMENTS

The authors gratefully acknowledge Granja Rezende S.A. for donations of the biological material, and also would like to thank Juliana Alves São Julião, Cícero Donizete Pereira and Guilherme Rocha Lino de Souza for their suggestions. The CAPES foundation has partially financed this research through the Special Training Program (PET) scholarship awarded to Fernando H. Biase.

Send correspondence to Fernando Henrique Biase. Universidade Federal de Uberlândia, Instituto de Genética e Bioquímica, Laboratório de Genética Molecular, Campus Umuarama, Bloco 2E sala 24, 38400-902 Uberlândia, MG, Brazil. E-mail: [e-mail].

References

• Akane A, Shiono H, Matsubara K, Nakamura H, Hasegawa M and Kagawa M (1993) Purification of Forensic Specimens for the Polimerase Chain Reaction (PCR) Analysis. J of Forensic Sci 38: 691-701.
• Atmadja DS, Tatsuno Y, Ueno Y and Nishimura A (1995) The effect of extraction methods, the kind of organs samples and the examination delay on the yields and typing. K. J Med Sci 41:197-211.
• Borges M (1997) Marcadores moleculares e seus efeitos sobre características quantitativas de bovinos de corte. MSc Dissertation, Universidade Federal de Uberlândia, Uberlândia.
• Hoy MA (1994) DNA Amplification by the Polymerase Chain Reaction: Molecular Biology Made Accessible. In: Hoy MA.Insect Molecular Genetics: An Introduction Principles and Applications, Academic Press, San Diego, pp 203-244.
• Junqueira LC and Carneiro J (1995) Histologia Básica. Guanabara Koogan S.A, Rio de Janeiro, 512 pp.
• Saiki RK (1990) Amplification of genomic DNA. In: Iinnis MA, Gelfand DH, Sninski JJ and White TJ. PCR Protocols: A Guide to Methods and Applications. Academic Press. San Diego, pp 13-20.
• Sambrook J, Fritschi EF and Maniatis T (1989) Molecular cloning: a laboratorymanual, Cold Spring Harbor Laboratory Press, New York.

Publication Dates

• Publication in this collection
  06 Dec 2002
• Date of issue
  2002

History

• Accepted
  01 Aug 2002
• Received
  31 July 2000