DNA stabilization is critical for maximizing performance of fecal DNA-based colorectal cancer tests.
We have developed a multitarget, fecal DNA screening assay that detects the presence of gene-specific mutations and long DNA fragments associated with colorectal cancer (CRC). We continue to investigate methods that may be used to optimize clinical sensitivity. The goals of this investigation are to establish how sample handling conditions affect the stability of DNA in stool, thereby potentially limiting clinical sensitivity, and to determine conditions to ameliorate DNA degradation. A study was run comparing paired sample aliquots. Quantitative PCR data for matched aliquots was used to determine first the effect of sample incubation on total recovery and integrity of DNA, then the effect of stabilization buffer addition to stool on recoverable DNA, and finally the impact of buffer addition on assay sensitivity. Comparison of quantitative PCR data for paired aliquots shows that the amount of recoverable human DNA is negatively affected by storing stool samples (N = 43) at room temperature for > or =36 hours (P = 0.0018). However, the addition of stabilization buffer leads to a significant increase in recovery of DNA (P = 0.010), compared with samples incubated without buffer. Whereas the DNA Integrity Assay (DIA) is found to be sensitive to DNA degradation (sensitivity was reduced by 82%; P = 0.0002), point mutation marker sensitivity is more refractory. Overall, DNA can be stabilized by addition of buffer to the sample, leading to increased assay sensitivity.
Olson
Jeff
J
Exact Sciences Corporation, Marlborough, MA 01752, USA.
Whitney
Duncan H
DH
Durkee
Kristine
K
Shuber
Anthony P
AP
D002021:Buffers; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification*; D005243:Feces / Q000737:chemistry*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013048:Specimen Handling
D002021:Buffers; D004273:DNA, Neoplasm