Effect of nucleic acid binding dyes on DNA extraction, amplification and STR typing

We report on the effects of six dyes used in the detection of DNA on the process of DNA extraction, amplification, and detection of STR loci. While dyes can be used to detect the presence of DNA, their use is restricted if they adversely affect subsequent DNA typing processes. DiamondTM Nucleic Acid Dye, GelGreenTM, GelRedTM, RedSafeTM, SYBRR Green I, and EvaGreenTM were evaluated in this study. The percentage of dye removed during the extraction process was determined to be: 70.3% for SYBRR Green I; 99.6% for RedSafeTM; 99.4% for EvaGreenTM; 52.7% for DiamondTM Dye; 50.6% for GelRedTM, and; could not be determined for GelGreenTM. It was then assumed that the amount of dye in the fluorescent quantification assay had no effect on the DNA signal. The presence of all six dyes was then reviewed for their effect on DNA extraction. The t-test showed no significant difference between the dyes and the control. These extracts were then STR profiled and all dyes and control produced full DNA profiles. STR loci in the presence of GelGreenTM at 1X concentration showed increased amplification products in comparison to the control samples. Full STR profiles were detected in the presence of EvaGreenTM (1X), although with reduced amplification products. RedSafeTM (1X), DiamondTM Dye (1X), and SYBRR Green I (1X) all exhibited varying degrees of locus drop-out with GelRedTM generating no loci at all. We provide recommendations for the best dye to visualize the presence of DNA profile as a biological stain and its subsequent amplification and detection.