Efficiencies were estimated by comparing the number of "copies in" to "copies out" with quantitative polymerase chain reaction (qPCR). The first method, "fishing" for DNA, retained only 9.06-3.53 percent (i.e., loss of 90.94-96.47 percent) of DNA targets ranging 108-288 base pairs (bps) in length. Minor improvement was achieved by using a modified fishing protocol, resulting in average retention of 31.41-12.08 percent of the same set of targeted molecules; however, of equal concern was the inability of the method to remove more non-target DNA molecules than targeted molecules. The second method, primer extension capture (PEC0 retained 15.88-2.24 percent (i..e., loss of 84.12-p7.86 percent) of the same target molecules. Experimental modifications of PEC sought to increase this efficiency without success. The third method, "mega-probe" capture, increased the count of target molecules from the same DNA standards by 702.46 percent, an impossible outcome. In observations of negative controls, bait molecules were counted as captured copies when, in fact, there were no copies to capture. Due to unexpected experimental outcomes, researchers were unable to estimate the efficiency of this method in its removal of non-target molecules. Researchers' principal concern about mega-probe, however, is the possibility that the mega-probe bait becomes counted as captured target molecules, such that any attempt to measure its efficiency in retaining targeted molecules will be biased. The losses of the molecules documented in this study are attributable to 1) the inefficiency of retaining molecules by purification using the Qiagen MinElute Purification Kit; 2) heat degradation of the DNA molecules, making them unavailable for PCR amplification; or 3) both. 11 tables, 2 figures, and 28 references
Bait and Capture: Holding on to Molecules of Interest
NCJ Number
251646
Date Published
July 2015
Length
96 pages
Annotation
This study's objective was to document the efficiency of three DNA bait-capture methods by measuring their ability to retain targeted DNA molecules and their ability to remove non-target DNA molecules.
Abstract
Date Published: July 1, 2015