Detection of colorectal cancer genes using a dye-free method combining barcode-base multiplex ligation-dependent probe amplification and pyrosequencing

Early detection of abnormal expression levels of cancer-related genes is crucial for reducing cancer mortality. Here, we describe a dye-free multiplex bioassay for simultaneous and quantitative analysis of the expression levels of multiple genes from one sample in a single assay, based on Sequence-tagged Multiplex ligationdependent probe Amplification (MLPA) coupled with pyrosequencing (termed as “SMAP”). Each pair of MLPA probes, containing a designed barcode, represents a gene of interest; thus, the use of various dyes to label different genes was avoided. The unique three-base barcode design on the probes, which can be decoded by pyrosequencing, enables individual quantification of the expression levels of six genes. Moreover, a new carryover contamination prevention system based on the use of restriction endonucleases was developed for PCR-based diagnostic screening assays. SMAP analysis revealed significant differences between the expression levels of CRC-related genes in the tumor tissues and normal tissues from a CRC patient. For PCR-based diagnostic screening assays, 0.5 U of the FokI restriction endonuclease was sufficient for the removal 0.01 pmol of PCR contamination. The ability to analyze the expression levels of a greater number of cancer-related genes would improve diagnostic sensitivity and efficacy. SMAP is amenable to the detection of an increased number of genes by lengthening the artificially designed barcodes; thus our method provides a promising means for cancer diagnostics and improving the treatment options available to cancer patients.