Pollen DNA Isolation Methods: Literature Review and Benefit of Adding Chloroform:Isoamyl Alcohol to Commercial DNA Kits

Successful isolation of pollen DNA is required for multiple disciplines. However, pollen DNA yields are typically low, perhaps because of physical-chemical challenges associated with lysing and exine removal. Here, an initial systematic literature review showed that efforts to improve pollen DNA isolation have focused primarily on optimizing lysis bead type and duration, and identifying effective commercial kits and/or in-house methods. Studies have not apparently focused on preventing clogging of kit-based DNA purification columns caused by post-lysis pollen debris including the exine. Here, chloroform:isoamyl alcohol 24:1 (C:I) was added as a post-lysis cleaning treatment to a commercial DNA isolation kit-based protocol to test its effect on pollen DNA yield of maize (Zea mays L., corn) as a model. C:I was tested in conjunction with different lysis durations and pollen quantities, along with lysis bead type, to determine optimal combinations. C:I improved final DNA yields ≤31% regardless of lysis duration and pollen quantity, compared to non-C:I controls. The optimal lysis duration depended on pollen quantity and vice versa, although bead type demonstrated a greater impact when coupled with C:I. The resulting DNA was of sufficient quality for microbiome analysis. This optimized protocol may minimize the pollen amount required for robust DNA isolation.