Incubation, pre-lysis and post-purification on the yield and purity of nucleic acids extracted from blood of domestic goats contained in FTA cards

Abstract

Molecular techniques require extractions of nucleic acids in adequate quantity and purity. This work describes a generalized linear model (GLM) of an adjusted factor with fixed effects on nucleic acid yield (ng/μl) and purity (A260/A280 and A260/A230), for five methods of DNA extraction using FTA cards with goat (Capra aegagrus hircus) blood. Two commercial methods based on silica columns (Invitrogen and Macherey Nagel; MN), the chelating resin method (Chelex), the CTAB method and the phenol-chloroform-isoamyl alcohol (PCI) method were tested. Additionally, for MN, an incubation step with PBS (Phosphate Buffered Saline) buffer at high temperature prior to lysis and a purification step post extraction were evaluated using a fixed-effect model of two factors with interaction. DNA concentrations and purity ratios were variable; the highest concentration was obtained with the MN kit (170.45 ng/μl), but with deficiencies in purity (0.32 of A260/A230, 0.34 of A260/A280). Despite this, all extraction methods generated PCR products with specific D-loop primers (mtDNA). The combined effect of the pre-incubation and post-purification stages yielded satisfactory purity values (1.89 for A260/A230 and 1.65 for A260/A280), as well as concentration ratios (476.78 ng/μl) with low variability. In conclusion, the concentration and purity of DNA from blood samples is greatly improved when using a commercial kit in combination with pre-lysis incubation and post-extraction purification. These nucleic acids are suggested for use in potential molecular applications a posteriori.