Digital detection of multiple minority mutants in stool DNA for noninvasive colorectal cancer diagnosis.
Somatic mutations in stool DNA are quite specific to colorectal cancer (CRC), but a method being able to detect the extraordinarily low amounts of mutants is challengeable in sensitivity. We proposed a hydrogel bead-array to digitally count CRC-specific mutants in stool at a low cost. At first, multiplex amplification of targets containing multiple mutation loci of interest is carried out by a target enriched multiplex PCR (Tem-PCR), yielding the templates qualified for emulsion PCR (emPCR). Then, after immobilizing the beads from emPCR on a glass surface, the incorporation of Cy3-dUTP into the mutant-specific probes, which are specifically hybridized with the amplified beads from emPCR, is used to color the beads coated with mutants. As all amplified beads are hybridized with the Cy5-labeled universal probe, a mutation rate is readily obtained by digitally counting the beads with different colors (yellow and red). A high specificity of the method is achieved by removing the mismatched probes in a bead-array with electrophoresis. The approach has been used to simultaneously detect 8 mutation loci within the APC, TP53, and KRAS genes in stools from eight CRC patients, and 50% of CRC patients were positively diagnosed; therefore, our method can be a potential tool for the noninvasive diagnosis of CRC.
Department of Pharmacology, Jinling Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu 210002, PR China.
Lili
L
Deng
Zongtai
Z
Qi
Binjie
B
Zou
Haiping
H
Wu
Huan
H
Huang
Tomoharu
T
Kajiyama
Hideki
H
Kambara
Guohua
G
Zhou
D002232:Carbocyanines / Q000032:analysis; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D004247:DNA / Q000032:analysis / Q000235:genetics*; D003856:Deoxyuracil Nucleotides / Q000032:analysis; D005243:Feces / Q000737:chemistry*; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D009154:Mutation; D059645:Mutation Rate; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
C088941:3'-deoxy-5-(cyanine dye 3)uridine 5'-trisphosphate; D002232:Carbocyanines; D003856:Deoxyuracil Nucleotides; C085321:cyanine dye 5; D004247:DNA