DNA bait capture uses synthetic biotinylated DNA primers to bind target DNA, which are then immobilized onto streptavidin coated magnetic beads and drawn to a magnet. Bound DNA should, therefore, be isolated from non-target DNA and impurities (e.g., PCR inhibitors) and can be later eluted from the beads for downstream applications. Efficiencies were estimated by comparing the number of "copies in" to "copies out" with quantitative polymerase chain reaction (qPCR). Retention of target DNA molecules, ranging from 109 to 288 base pairs (bps) in length, averaged just 9.06-3.53 percent (i.e., loss of 90.94-96.47 percent) using the fishing protocol as originally described. Some improvement was achieved by employing a modified protocol (i.e., with a shortened hybridization time, use of twice the amount of M-270 streptavidin-coated beads, and modified bead washing), resulting in average retention of 31.41-12.08 percent of the same set of targeted molecules. Noted was the lack of efficacy in removing non-target DNA molecules as opposed to targeted molecules. It was also observed that most of the molecules (61.35-69.49 percent) are "lost" during the essential hybridization step of the fishing protocol, suggesting its suitability for high copy number samples only. Although the bait capture method may be useful in the study of polymerase chain reaction (PCR) inhibited DNA samples as previously suggested, it is necessary to carefully weigh this possible advantage against the degree of expected DNA loss and the non-selectivity of the method for targeted over non-targeted DNA. (publisher abstract modified)
Downloads
Similar Publications
- Development of Microscopical Methods for the Systematic Analysis of Chemically Reacted, Improvised Low Explosives and Related Residues
- Development of a Comprehensive Workflow for Postmortem Insulin Testing in Cases of Suspected Insulin Homicide
- Workflow to Facilitate the Detection of New Psychoactive Substances and Drugs of Abuse in Influent Urban Wastewater