An electrophoretic capture method for efficient recovery of rare sequences from heterogeneous DNA.
It is difficult to isolate rare, PCR-quality DNA from specimens containing large quantities of nonspecific DNA from multiple sources (heterogeneous DNA). Extracting human DNA from stool for colorectal cancer (CRC) screening tests exemplifies this technically challenging sample preparation problem. The stool matrix is complex, the DNA composition heterogeneous, and CRC-associated mutated DNA is rare. This report describes a novel solid phase DNA sequence-specific hybrid capture sample preparation method: the reversible electrophoretic capture affinity protocol (RECAP). We show that RECAP, compared with other methods, is capable of extracting linearly increasing amounts of human DNA from increasing amounts of total stool DNA in a manner that avoids co-purifying PCR inhibitors. RECAP thereby increases the yield of rare mutated DNA molecules and thus increases the detection sensitivity for CRC-associated mutations.
Kent Moore
J
J
EXACT Sciences Corporation, Marlborough, MA 01752, USA. kmoore@exactsciences.com
Smith
Jean A
JA
Whitney
Duncan H
DH
Durkee
Kristine H
KH
Shuber
Anthony P
AP
D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics* / Q000302:isolation & purification*; D004586:Electrophoresis / Q000379:methods*; D005243:Feces / Q000737:chemistry*; D006801:Humans; D017422:Sequence Analysis, DNA / Q000379:methods*; D013048:Specimen Handling / Q000379:methods*
D004273:DNA, Neoplasm