Image differentiated multiplex assays for multiplex detection of DNA mutations

A method for detecting the presence of DNA mutations in the KRAS, BRAF, CTNNB1, and APC genes, the method comprising:

• (a) isolating DNA from a sample;
• (b) amplifying the isolated DNA by polymerase chain reaction (PCR) using primer pairs specific for the loci of one or more DNA mutations in each of the KRAS, BRAF, CTNNB1, and APC genes;
• (c) hybridizing the amplified DNA with at least four probes, said at least four probes comprising one or more probes specific for a DNA mutation in each of the KRAS, BRAF, CTNNB1, and APC genes, wherein each of said at least four probes is coupled to a microcarrier, wherein each of the microcarriers comprises an identifier corresponding to the probe coupled thereto, and wherein each of the microcarriers comprises:
• (1) a substantially transparent polymer layer having a first surface and a second surface, the first and the second surfaces being parallel to each other,
• (2) a substantially non-transparent polymer layer, wherein the substantially non-transparent polymer layer is affixed to the first surface of the substantially transparent polymer layer and encloses a center portion of the substantially transparent polymer layer, and wherein the substantially non-transparent polymer layer comprises a two-dimensional shape representing an analog code, wherein the analog code represents the identifier, and
• (3) the probe specific for the DNA mutation, wherein the probe is coupled to at least one of the first surface and the second surface of the substantially transparent polymer layer in at least the center portion of the substantially transparent polymer layer;
• (d) detecting presence or absence of hybridization of the amplified DNA with said at least four probes, wherein hybridization between the amplified DNA and one of the probes indicates the presence of the DNA mutation corresponding to the probe;
• (e) detecting the identifiers of the microcarriers;
• and
• (f) correlating the detected identifiers of the microcarriers with the detected presence or absence of hybridization of the amplified DNA to the corresponding probes of the microcarriers.