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An electrophoretic capture method for efficient recovery of rare sequences from heterogeneous DNA J. Kent Moore, Jean A. Smith, Duncan H. Whitney, Kristine H. Durkee, Anthony P. Shuber
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Despite the increase in DNA yield, end-point PCR using these samples showed very little human APC sequence amplification ((Figure 3)A). Results are shown for cancer stool sample 02-8854, but were similar for the other stools tested. Faint PCR bands were seen only at high dilutions of maxi-column purified DNA. Similarly, real-time PCR quantification detected little to no APC sequence and again only at high dilutions of maxi-column purified DNA. Robust PCR amplification of the APC sequence when additional human DNA is spiked into the eluted stool DNA demonstrated that the columns successfully purified the total stool DNA of PCR inhibitors ((Figure 3)B). However, in samples with the highest DNA concentration, spiked DNA amplifications were still inhibited. This indicates that PCR amplification can become inhibited with excessive amounts of total stool DNA, possibly because of aberrant priming on the large quantities of nontarget DNA. Repeating the scale-up using fresh stools homogenized for DNA isolation as described in the manufacturer's protocol for large amounts of stool showed similar poor results. Figure 3. End-point agarose gel analysis of APC PCR amplification using stool DNA derived from commercial purification kits. Agarose gel pictures of PCR amplifications for the APC sequence using stool DNA generated from increasing sample volumes applied to commercial stool DNA purification kits (Qiagen). (A) Amplification using 10 µL of undiluted purified DNA from stool sample 02–8854. Also shown are PCR amplifications using undiluted purified DNA and 2-fold serial dilutions recovered from the QIAamp maxi-column. Similar results were found using purified DNA from stool samples GP-164 and 02-5860 (PCR Control, 7 GE-purified human DNA in 2 µg purified E. coli DNA). (B) Results from purified DNA from stool sample 02–8854 spiked with human DNA at 1250 GE per PCR is also illustrated. (PCR Control, 1250 GE-purified human DNA spike with no stool sample added.) GE, genomic equivalents. We have shown in this report that sequence-specific hybridization capture methods are more efficient in the recovery and subsequent amplification of rare DNA molecules from heterogeneous DNA samples than methods that purify the total DNA. The removal of extraneous DNA and thus the enrichment of target leads to more robust PCR. The RECAP electrophoretic hybridization capture process is especially suited for minimally purified complex heterogeneous DNA samples. We have demonstrated that this process can successfully recover rare DNA molecules from both small (0.2 g) and large (8 g) initial quantities of stool samples. The magnetic bead-based hybridization capture method has been used to effectively recover human DNA from stool (21,22,23). However, as shown in this report and in previous unpublished studies (EXACT Sciences), large stool volumes are often associated with incomplete removal of PCR inhibitors, compromising downstream PCR reactions. While the addition of post-capture purification steps can address this issue, it would add extra time and expense to the magnetic bead-based process. This extra effort required could be offset with the ease of adapting the magnetic bead processing to high-throughput automation. As the Ultralink beads used in this study have a very high binding capacity, additional capture probes can be incorporated in a multiplex format to simultaneously capture multiple DNA targets, greatly increasing RECAP's versatility. Currently, we are validating RECAP with a blinded set of clinical stool samples for a multiplex capture of several cancer-related human gene targets. Although developed for human DNA capture in stool, RECAP is a generic platform technology whose specificity is only dependent on the bound capture probes. RECAP could be applied to other biological samples looking for rare DNA targets in a background of larger amounts of heterogeneous DNA molecules and where PCR inhibitors are also an issue. RECAP could also be optimized for RNA or protein capture by attaching the appropriate ligand to the streptavidin beads. Acknowledgments J.K.M. and J.A.S. have contributed equally to this work. We would like to thank John Millholland and Barry M. Berger for their critical review of the manuscript. A special thanks to Patty David for her help in the development of RECAP. Competing Interests Statement J.K.M., J.A.S., and K.H.D. are employees of EXACT Sciences Corporation and are all shareholders of the company. D.H.W. and A.P.S. were formerly employed by EXACT Sciences and performed this work while employed there. 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