Oligonucleotides and methods for detecting KRAS and PIK3CA mutations

#The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 4, 2014, is named CorrectedSL 2009P14771US 090814.txt and is 12 kilobytes in size.

#The present invention relates generally to oligonucleotides and methods for detecting KRAS and PIK3CA mutations in patient samples. More specifically, the present invention relates to primers and kPCR assays that are capable of detecting KRAS codon 12, 13, and 61 mutations and PIK3CA codon 542, 545, and 1047 mutations with high specificity and sensitivity.

#Tyrosine kinase inhibitors (TKIs) are a class of anti-cancerous monoclonal antibody drugs that target tumor growth, such as metastasized colorectal cancers (mCRC). TKIs are designed to block receptors and stop the growth signals that fuel the tumor thereby stopping the growth of the cancer cells.

#FIG. 1 shows the oligonucleotide map for the KRAS gene, including exon 2. FIG. 1 discloses SEQ ID NO. 34.

#FIG. 2A shows KRAS codon 12 and 13 sequences and locations on Accession #NT—009714.16/Hs12—9871. FIG. 2A discloses SEQ ID NOs. 1-12.

#FIG. 2B shows KRAS codon 61 sequences and locations on Accession #NT—009714.16/Hs12—9871. FIG. 2B discloses SEQ ID NOs. 23-26.

#Set forth below is a description of what are currently believed to be preferred embodiments of the claimed invention. Any alternates or modifications in function, purpose, or structure are intended to be covered by the claims of this application. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

#The terms “kinetic PCR” (kPCR) and “realtime PCR” are used interchangeably herein to refer to the detection of polymerase chain reaction (PCR) products via a fluorescent signal generated by the coupling of a fluorogenic dye molecule and a quencher moiety to the same or different oligonucleotide substrates. Examples of commonly used probes used for kPCR assay include the following probes: TAQMAN® probes, Molecular Beacons probes, SCORPIONS® probes, and SYBR® Green probes. TAQMAN®, Molecular Beacons, and SCORPIONS® probes each have a fluorescent reporter dye (also called a “fluor”) attached to the 5′ end of the probes and a quencher moiety coupled to the 3′ end of the probes. TAQMAN® probes are designed to hybridize to an internal region of a PCR product. In the unhybridized state, the proximity of the fluor and the quench molecules prevents the detection of fluorescent signal from the probe; during PCR, when the polymerase replicates a template on which a TAQMAN® probe is bound, the 5′-nuclease activity of the polymerase cleaves the probe thus, increasing fluorescence with each replication cycle. Unlike TAQMAN® probes, Molecular Beacons, which form a stem-loop structure when free in solution, remain intact during the amplification reaction. Molecular Beacons fluoresce during hybridization when the fluorescent dye and the quencher are separated. For signal measurement to be effective, the fluor and quencher must rebind in every cycle. SCORPIONS® probes, which maintain a stem-loop configuration in the unhybridized state, has at its 3′ end an additional sequence that is complementary to the extension product of the primer that is linked to the 5′ end of a specific primer via a non-amplifiable monomer. After extension of the SCORPIONS® primer, the specific probe sequence is able to bind to its complement within the extended amplicon thus opening up the hairpin loop such that the fluor is no longer quenched and signal is seen. SYBR® Green probes binds double-stranded DNA and upon excitation emit light; thus as PCR product accumulates, fluorescence increases.

#The term “singleplex” refers to a single assay that is not carried out simultaneously with any other assays. For example, within a multiwell plate, a singleplex assay refers to a single reaction that is performed within a single well of the multiwall plate. Singleplex assays include individual assays that are carried out sequentially.