African Journal of Biotechnology Vol. 6 (4), pp. 481-486, 19 February 2007   

ISSN 1684–5315 © 2007 Academic Journals        

Full Length Research Paper

Optimizing factors influencing DNA extraction from fresh whole avian blood

H. Khosravinia^1*, H. N. Narasimha Murthy², D. Thertha Parasad³ and N. Pirany⁴

¹Department of Animal Science, Agriculture Faculty, Lorestan University, P.B. 465, Khoramabad-68135, Lorestan, Iran.

²Department of Poultry Science, Veterinary College, UAS, Bangalore-560024, India.

³Department of Biothechnology, University of Agricultural Sciences, GKVK, Bangalore, 560065. India.

⁴Department of Animal Science, Faculty of Agriculture, University of Tabriz, Tabriz, Iran.

*Corresponding author email: khosravi_fafa@yahoo.com

Accepted 20 November, 2006

A study was conducted to optimize the efficient combination of lysis buffer, proteinase K, incubation time, phenol-chloroform-isoamyl alcohol (PCI) volume, spinning rate (rpm), and precipitation agent on quantity and quality of DNA extracted from various volumes of avian blood. Blood samples were collected in EDTA and swiftly transferred to a laboratory for DNA extraction. The lysis buffer used had composition 5 M NaCl, 1 M Tris (pH=8.0), 0.5 M EDTA and20% SDS. The effect of various levels for each factor concerned was examined using General Linear Models or t-test procedures of SAS^® software. The volume removed from the top aqueous part following the first and the second PCI washings was included in the models as a continuous variable; the variables of interest were OD₂₈₀, OD₂₆₀, OD₂₆₀/OD₂₈₀ (as quality criterion), total extracted DNA, extraction efficiency (µg DNA/µl blood), assay scores for easiness of removing the top aqueous phase after the first (assay 1) and the second (assay 2) spinning. The optimum level of factors significant for DNA extraction from fresh avian blood was found to be lysis buffer : blood sample ratio of 31:36 (µl : µl), incubation time of 60-70 min at 58^oC, two washings with PCI at 1.2:1.3 PCI : top aqueous phase (µl : µl) for the first and 1.4 for the second washing, centrifuge of homogenised sample at 2000 - 2500 rpm for 20 min, precipitation of DNA with 1.5 – 2.0 volume of absolute ethanol.

Key words: DNA extraction, phenol-chloroform, avian, whole blood.