American Association for Cancer Research
19406207capr130145-sup-tab_1_pdf67k.pdf (65.81 kB)

Supplementary Table 1 from Ultrasensitive Detection of Unknown Colon Cancer-Initiating Mutations Using the Example of the Adenomatous Polyposis Coli Gene

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journal contribution
posted on 2023-04-03, 19:21 authored by Christian Gerecke, Conny Mascher, Uwe Gottschalk, Burkhard Kleuser, Bettina Scholtka

PDF - 67K, LNA blocker sequences and reaction conditions for use in Wild-type Blocking (WTB) PCR of selected APC and KRAS segments.



Detection of cancer precursors contributes to cancer prevention, for example, in the case of colorectal cancer. To record more patients early, ultrasensitive methods are required for the purpose of noninvasive precursor detection in body fluids. Our aim was to develop a method for enrichment and detection of known as well as unknown driver mutations in the Adenomatous polyposis coli (APC) gene. By coupled wild-type blocking (WTB) PCR and high-resolution melting (HRM), referred to as WTB-HRM, a minimum detection limit of 0.01% mutant in excess wild-type was achieved according to as little as 1 pg mutated DNA in the assay. The technique was applied to 80 tissue samples from patients with colorectal cancer (n = 17), adenomas (n = 50), serrated lesions (n = 8), and normal mucosa (n = 5). Any kind of known and unknown APC mutations (deletions, insertions, and base exchanges) being situated inside the mutation cluster region was distinguishable from wild-type DNA. Furthermore, by WTB-HRM, nearly twice as many carcinomas and 1.5 times more precursor lesions were identified to be mutated in APC, as compared with direct sequencing. By analyzing 31 associated stool DNA specimens all but one of the APC mutations could be recovered. Transferability of the WTB-HRM method to other genes was proven using the example of KRAS mutation analysis. In summary, WTB-HRM is a new approach for ultrasensitive detection of cancer-initiating mutations. In this sense, it appears especially applicable for noninvasive detection of colon cancer precursors in body fluids with excess wild-type DNA like stool. Cancer Prev Res; 6(9); 898–907. ©2013 AACR.