Patent Publication Number: US-2018051325-A1

Title: Multiplex allele specific pcr assays for detection of estrogen receptor esr1 mutations

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     The present application claims priority to U.S. Provisional Application 62/376,799, filed 18 Aug. 2016, the disclosure of which is incorporated herein in its entirety. 
    
    
     SEQUENCE LISTING 
     The present application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on Jul. 10, 2017, is named 33815-US1_SL.txt and is 137,578 bytes in size. 
     BACKGROUND OF THE INVENTION 
     Seventy percent of breast cancers are estrogen receptor (ER) positive and, while the majority of these patients initially respond to hormone therapy, approximately 20-30% will become therapy refractory. Recent data suggest activating mutations in the estrogen receptor gene (ESR1) which are acquired during anti-estrogen treatment and rarely found in primary untreated ER positive breast cancer are associated with resistance. A growing number of activating ESR1 mutations located in the ligand-binding domain have been identified in samples from hormone-refractory breast cancer, the most common mutations include K303R, E380Q, V392I, S463P, K531E, V534E, P535H, L536Q/R, Y537S/N/C, D538G and R555C. While the mechanism of action is not fully elucidated for all mutations, it has been shown by in vitro experiments that mutations in L536Q, Y537S/C/N and D538G stabilize the estrogen receptor ligand binding domain in an active conformation, thus allowing recruitment of transcriptional coactivators in the absence of ligand. Hypersensitivity to estrogen is considered the mechanism for the K303R mutation. 
     Detection of ESR1 mutations thus has potential for predicting hormone resistance and directing therapy. Next generation sequencing (NGS) is a common approach for such detection, since it enables the simultaneous detection of many mutations with small amounts of samples. However, NGS is very labor-intensive, lengthy and expensive. Digital PCR (dPCR) represents a highly sensitive method that has been employed for ESR1 mutation detection, but it has a number of drawbacks: the dPCR workflow is lengthy and requires special equipment, and multiplexing is limited by the number of optical channels available on current dPCR instruments. 
     SUMMARY OF THE INVENTION 
     Provided herein are kits, assays, and methods for detecting mutations in the ESR1 gene, and methods of treatment for individuals with hormone sensitive cancers. 
     Provided herein are kits comprising one or more vessels, each of the vessels holding two or more primer pairs, each primer pair specific for a different sequence in the ESR1 gene; one or more probes specific for a different sequence in the ESR1 gene, wherein each probe is labeled; a primer pair specific for an internal control sequence; and a probe specific for the internal control sequence. In some embodiments, the each probe is labeled with a fluorophore and quencher. In some embodiments, the kit comprises 1-10 vessels, e.g., 2-5, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more vessels. In some embodiments, each of the vessels holds two or more primer pairs (including the internal control primer pair), e.g., 3-16, 4-10, or 5-8. For example, all exon 8 mutations, internal control, and two or more other mutation primer pairs can be included in a single tube. In some embodiments, each of the vessels holds three to four probes (including the internal control probe). In some embodiments, each vessel further holds a thermostable DNA polymerase. 
     In some embodiments, the kit further includes positive controls, e.g., samples with known ESR1 mutations, and/or a negative control, e.g., a sample that does not include ESR1 mutations. 
     In some embodiments, each different sequence of the ESR1 gene is selected from the group consisting of comprising ESR1 V422DelV, ESR1 S463P, ESR1 L536H, ESR1 L536P, ESR1 L536Q, ESR1 L536R, ESR1 D538G, ESR1 K303R, ESR1 E380Q, ESR1 L536_D538&gt;P, ESR1 Y537C, ESR1 Y537N, ESR1 Y537S, ESR1 S341L, ESR1 L429V, ESR1 V533M, ESR1 V534E, and ESR1 P535H. In some embodiments the kit includes at least two primer pairs selected from the group consisting of a primer pair specific for ESR1 V422DelV, a primer pair specific for ESR1 S463P, a primer pair specific for ESR1 L536H, a primer pair specific for ESR1 L536P, a primer pair specific for ESR1 L536Q, a primer pair specific for ESR1 L536R, a primer pair specific for ESR1 D538G, ESR1 K303R, a primer pair specific for ESR1 E380Q, a primer pair specific for ESR1 L536_D538&gt;P, a primer pair specific for ESR1 Y537C, a primer pair specific for ESR1 Y537N, a primer pair specific for ESR1 Y537S, a primer pair specific for ESR1 S341L, a primer pair specific for ESR1 L429V, a primer pair specific for ESR1 V533M, a primer pair specific for ESR1 V534E, and a primer pair specific for ESR1 P535H. In some embodiments, at least one primer includes a modified, non-naturally occurring nucleotide. 
     In some embodiments, the kit comprises a primer pair specific for ESR1 V422DelV, a primer pair specific for ESR1 S463P, a primer pair specific for ESR1 L536H, a primer pair specific for ESR1 L536P, a primer pair specific for ESR1 L536Q, a primer pair specific for ESR1 L536R, a primer pair specific for ESR1 D538G, ESR1 K303R, a primer pair specific for ESR1 E380Q, a primer pair specific for ESR1 L536_D538&gt;P, a primer pair specific for ESR1 Y537C, a primer pair specific for ESR1 Y537N, a primer pair specific for ESR1 Y537S, a primer pair specific for ESR1 S341L, a primer pair specific for ESR1 L429V, a primer pair specific for ESR1 V533M, a primer pair specific for ESR1 V534E, and a primer pair specific for ESR1 P535H. In some embodiments, the kit further comprises a probe that specifically detects ESR1 V422DelV, ESR1 S463P, ESR1 L536H, ESR1 L536P, ESR1 L536Q, ESR1 L536R, ESR1 D538G, ESR1 K303R, ESR1 E380Q, ESR1 L536_D538&gt;P, ESR1 Y537C, ESR1 Y537N, ESR1 Y537S, ESR1 S341L, ESR1 L429V, ESR1 V533M, ESR1 V534E, and ESR1 P535H. 
     In some embodiments, the kit comprises a (i) a first vessel holding a primer pair specific for ESR1 V422DelV, a primer pair specific for ESR1 S463P, a primer pair specific for ESR1 L536H, a primer pair specific for ESR1 L536P, a primer pair specific for ESR1 L536Q, a primer pair specific for L536R, and a primer pair specific for ESR1 D538G; (ii) a second vessel holding a primer pair specific for ESR1 K303R, a primer pair specific for ESR1 E380Q, a primer pair specific for ESR1 L536_D538&gt;P, a primer pair specific for ESR1 Y537C, a primer pair specific for ESR1 Y537N, and a primer pair specific for ESR1 Y537S; and (iii) a third vessel holding a primer pair specific for ESR1 S341L, a primer pair specific for ESR1 L429V, a primer pair specific for ESR1 V533M, a primer pair specific for ESR1 V534E, and a primer pair specific for ESR1 P535H. In some embodiments, the first vessel further holds probes specific for ESR1 V422DelV, ESR1 S463P, ESR1 L536H, ESR1 L536P, ESR1 L536Q, ESR1 L536R, and ESR1 D538G. In some embodiments, a single probe specifically detects ESR1 L536H, ESR1 L536P, ESR1 L536Q, ESR1 L536R, and ESR1 D538G. In some embodiments, the second vessel further holds probes specific for ESR1 K303R, ESR1 E380Q, ESR1 L536_D538&gt;P, ESR1 ESR1 Y537C, ESR Y537N, ESR1 Y537S. In some embodiments, a single probe specifically detects ESR1 L536_D538&gt;P, ESR1 ESR1 Y537C, ESR Y537N, ESR1 Y537S. In some embodiments, the third vessel holds probes specific for ESR1 S341L, ESR1 L429V, ESR1 V533M, ESR1 V534E, and ESR1 P535H. In some embodiments, a single probe specifically detects ESR1 V533M, ESR1 V534E, and ESR1 P535H. 
     In some embodiments, the allele-specific primer to detect the ESR1 K303R mutation is selected from the group consisting of SEQ ID NOs: 479, 481, and 484, and in some embodiments, the common primer and probe sequences are SEQ ID NOs:476 and 477, respectively. In some embodiments, the allele-specific primer to detect the ESR1 S341L mutation is selected from the group consisting of SEQ ID NOs:239, 240, 242, 245, 246, 247, 253, 254, 255, 256, 257, 258, and 259, and in some embodiments, the common primer and probe sequences are SEQ ID NOs:235 and 237, respectively. In some embodiments, the allele-specific primer to detect the ESR1 E380Q mutation is selected from the group consisting of SEQ ID NOs:32, 34, 36, 37, 38, 39, 40, 41, and 42, and in some embodiments, the common primer and probe sequences are SEQ ID NOs:528 and 31, respectively. In some embodiments, the allele-specific primer to detect the ESR1 V422DELV mutation is selected from the group consisting of SEQ ID NOs:287, 294, 295, 296, 297, 298, and in some embodiments, the common primer and probe sequences are SEQ ID NOs:285 and 286, respectively. In some embodiments, the allele-specific primer to detect the ESR1 L429V mutation is selected from the group consisting of SEQ ID NOs:66, 68, 69, 70, 72, 73, 75, 76, 77, 778, 79, 80, and 81, and in some embodiments, the common primer and probe sequences are SEQ ID NOs:59 and 60, respectively. In some embodiments, the allele-specific primer to detect the ESR1 S463P mutation is selected from the group consisting of SEQ ID NOs:264, 265, 267, 268, 269, 270, 271, 272, 281, 282, 283, and 275, and in some embodiments, the common primer and probe sequences are SEQ ID NOs:261 and 262, respectively. 
     In some embodiments, a common primer and probe are used to amplify and detect all ESR1 exon 8 mutations in the assay, e.g., SEQ ID NOs:314 and/ or 394, and 407, respectively. In some embodiments, the allele-specific primer to detect the ESR1 V533M mutation is selected from the group consisting of SEQ ID NOs:322, 329, 330, 345, 346, 347, 348, 349, 350, 352, and 353. In some embodiments, the allele-specific primer to detect the ESR1 V534E mutation is selected from the group consisting of SEQ ID NOs:364, 365, 367, 368, 370, 371, 373, 378, 388, 391, 392, and 393. In some embodiments, the allele-specific primer to detect the ESR1 P535H mutation is selected from the group consisting of SEQ ID NOs:181, 188, 189, 190, 191, 198, 199, 200, 203, 204, 205, 208, 209, 212, 225, 226, 228, 229, and 231. In some embodiments, the allele-specific primer to detect the ESR1 L536H mutation is selected from the group consisting of SEQ ID NOs:96, 101, 104, 105, 107, 110, 111, 112, 113, 114, 115, 116, and 117. In some embodiments, the allele-specific primer to detect the ESR1 L536P mutation is selected from the group consisting of SEQ ID NOs:134, 135, and 137. In some embodiments, the allele-specific primer to detect the ESR1 L536Q mutation is selected from the group consisting of SEQ ID NOs:141, 151, 154, 155, 157, 158, 159, and 160. In some embodiments, the allele-specific primer to detect the ESR1 L536R mutation is selected from the group consisting of SEQ ID NOs:161, 172, 174, 175, 176, 177, 178, 179, and 180. In some embodiments, the allele-specific primer to detect the ESR1 Y537C mutation is selected from the group consisting of SEQ ID NOs:397, 408, 415, 416, 417, 418, 419, 420, and 424. In some embodiments, the allele-specific primer to detect the ESR1 Y537N mutation is selected from the group consisting of SEQ ID NOs:426, 436, 441, 445, 446, 447, and 448. In some embodiments, the allele-specific primer to detect the ESR1 Y537S mutation is selected from the group consisting of SEQ ID NOs:449, 459, 466, 467, 468, 469, 470, 471, and 472. In some embodiments, the allele-specific primer to detect the ESR1 D538G mutation is selected from the group consisting of SEQ ID NOs: 14, 21, 22, 23, 24, and 25. In some embodiments, the allele-specific primer to detect the ESR1 L536_D538&gt;P mutation is selected from the group consisting of SEQ ID NOs:84, 85, 86, 87, 88, 89, 90, 91, 92, and 93. 
     In some embodiments, the kit includes oligonucleotides having the sequences of SEQ ID NOs:24, 113, 134, 158, 178, 264, 261, 262, 285, 286, 298, 314, 394, 407, 31, 42, 90, 415, 447, 469, 476, 477, 481, 528, 59, 60, 70, 231, 235, 237, 245, 350, and 388. In some embodiments, each vessel includes an internal control, e.g., SEQ ID NOs:511, 514, and 517. 
     Further provided are methods for determining (e.g., detecting) the presence or absence of two or more ESR1 mutations in a sample from an individual, comprising (i) obtaining a sample from the individual; (ii) carrying out multiplex allele-specific PCR to determine the presence or absence of two or more ESR1 mutations. In some embodiments, the sample is selected from blood, plasma, serum, urine, or mucosal tissue (e.g., from a buccal swab). In some embodiments, the individual has, or was diagnosed with, hormone responsive cancer (e.g., estrogen receptor and/or progesterone receptor positive breast or ovarian cancer). In some embodiments, the individual is undergoing hormone therapy. In some embodiments, the method further comprises providing hormone therapy to the individual prior to step (i) (e.g., treatment with a SERM, aromatase inhibitor, or LH blocking agent). In some embodiments, the method further comprises providing modified treatment to the individual when the presence of an ESR1 mutation is determined in step (ii) (e.g., an additional hormone therapy, or standard chemotherapy). 
     In some embodiments, the method comprises carrying out multiplex allele-specific PCR to determine the presence or absence of ten or more ESR1 mutations, e.g., 10-20, 11, 12, 13, 14, 15, 16, 17, 18, or 19. In some embodiments, the two or more ESR1 mutations are selected from ESR1 V422DelV, ESR1 S463P, ESR1 L536H, ESR1 L536P, ESR1 L536Q, ESR1 L536R, ESR1 D538G, ESR1 K303R, ESR1 E380Q, ESR1 L536_D538&gt;P, ESR1 Y537C, ESR1 Y537N, ESR1 Y537S, ESR1 S341L, ESR1 L429V, ESR1 V533M, ESR1 V534E, and ESR1 P535H. In some embodiments, the two or more ESR1 mutations include ESR1 V422DelV, ESR1 S463P, ESR1 L536H, ESR1 L536P, ESR1 L536Q, ESR1 L536R, ESR1 D538G, ESR1 K303R, ESR1 E380Q, ESR1 L536_D538&gt;P, ESR1 Y537C, ESR1 Y537N, ESR1 Y537S, ESR1 S341L, ESR1 L429V, ESR1 V533M, ESR1 V534E, and ESR1 P535H. 
     In some embodiments, the multiplex allele-specific PCR is carried out using a kit as described herein, e.g., comprising 1-10 vessels, e.g., 2-5, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more vessels. In some embodiments, the kit comprises three vessels. For example, the allele-specific multiplex PCR can be carried out in three vessels such that (i) ESR1 V422DelV, ESR1 S463P, ESR1 L536H, ESR1 L536P, ESR1 L536Q, L536R, and ESR1 D538G are detected in a first vessel; (ii) ESR1 K303R, ESR1 E380Q, ESR1 L536_D538&gt;P, ESR1 Y537C, ESR1 Y537N, and ESR1 Y537S are detected in a second vessel; and (iii) ESR1 S341L, ESR1 L429V, ESR1 V533M, ESR1 V534E, and ESR1 P535H are detected in a third vessel. 
     Further provided herein are methods of providing modified treatment of an individual with a hormone responsive cancer (e.g., breast cancer) that is undergoing hormone therapy (e.g., SERM, aromatase inhibitors, and/or lutenizing hormone blockers). In some embodiments, the method comprises (i) obtaining a sample from the individual (e.g., blood, plasma, serum, urine, tissue, FFPET, etc.); (ii) carrying out multiplex allele-specific PCR to determine the presence or absence of two or more ESR1 mutations; and (iii) providing modified treatment of the individual if the presence of an ESR1 mutation is determined. In some embodiments, step (ii) is carried out using a kit as described herein. 
     In some embodiments, the multiplex allele-specific PCR determines the presence or absence of ten or more ESR1 mutations. In some embodiments, the two or more (or ten or more) ESR1 mutations are selected from the group consisting of ESR1 V422DelV, ESR1 S463P, ESR1 L536H, ESR1 L536P, ESR1 L536Q, ESR1 L536R, ESR1 D538G, ESR1 K303R, ESR1 E380Q, ESR1 L536_D538&gt;P, ESR1 Y537C, ESR1 Y537N, ESR1 Y537S, ESR1 S341L, ESR1 L429V, ESR1 V533M, ESR1 V534E, and ESR1 P535H. In some embodiments, the presence or absence of ESR1 V422DelV, ESR1 S463P, ESR1 L536H, ESR1 L536P, ESR1 L536Q, ESR1 L536R, ESR1 D538G, ESR1 K303R, ESR1 E380Q, ESR1 L536_D538&gt;P, ESR1 Y537C, ESR1 Y537N, ESR1 Y537S, ESR1 S341L, ESR1 L429V, ESR1 V533M, ESR1 V534E, and ESR1 P535H is determined. 
     In some embodiments, steps (i)-(iii) are carried out 0.5 to 5 years after the individual starts taking hormone therapy. In some embodiments, the method is carried out more than once during hormone therapy, e.g., to monitor potential resistance of the tumor to hormone therapy. In some embodiments, the method is carried out periodically, e.g., every 6 months, while in some embodiments, the method is carried out upon clinical progression in the individual (e.g., tumor growth, reduced response to hormone therapy, etc.). In some embodiments, step (iii) comprises providing an additional hormone therapy or standard chemotherapy to the individual. 
     In addition, provided herein are methods of treating an individual with a hormone responsive cancer (e.g., breast or ovarian cancer). In some embodiments, the method comprises (i) providing hormone therapy to the individual; (ii) obtaining a sample from the individual; (iii) carrying out multiplex allele-specific PCR to determine the presence or absence of two or more ESR1 mutations; and (iv) providing modified treatment to the individual if the presence of an ESR1 mutation is determined. In some embodiments, the hormone therapy is SERM. 
     In some embodiments, the multiplex allele-specific PCR determines the presence or absence of ten or more ESR1 mutations. In some embodiments, the two or more (or ten or more) ESR1 mutations are selected from the group consisting of ESR1 V422DelV, ESR1 S463P, ESR1 L536H, ESR1 L536P, ESR1 L536Q, ESR1 L536R, ESR1 D538G, ESR1 K303R, ESR1 E380Q, ESR1 L536_D538&gt;P, ESR1 Y537C, ESR1 Y537N, ESR1 Y537S, ESR1 S341L, ESR1 L429V, ESR1 V533M, ESR1 V534E, and ESR1 P535H. In some embodiments, the presence or absence of ESR1 V422DelV, ESR1 S463P, ESR1 L536H, ESR1 L536P, ESR1 L536Q, ESR1 L536R, ESR1 D538G, ESR1 K303R, ESR1 E380Q, ESR1 L536_D538&gt;P, ESR1 Y537C, ESR1 Y537N, ESR1 Y537S, ESR1 S341L, ESR1 L429V, ESR1 V533M, ESR1 V534E, and ESR1 P535H is determined. 
     In some embodiments, steps (ii)-(iii) are carried out 0.5-5 years after step (i) (after the individual starts taking hormone therapy). In some embodiments, steps (ii)-(iii) are carried out more than once during hormone therapy. In some embodiments, the method is carried out periodically, e.g., every 6 months, while in some embodiments, the method is carried out upon clinical progression in the individual (e.g., tumor growth, reduced response to hormone therapy, etc.). In some embodiments, step (iv) comprises providing an additional hormone therapy (e.g., aromatase inhibitor and/or lutenizing hormone blocker) or standard chemotherapy to the individual. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1A ,  FIG. 1B , and  FIG. 1C  compare discrimination of primers and probes for detecting mutant and wild type samples.  FIG. 1A  shows results (amplification growth curves) using primers and a probe designed to be specific for K303R that shows poor discrimination between wild type and mutant.  FIG. 1B  shows results using primers and a probe designed to be specific for K303R that shows better discrimination between wild type and mutant.  FIG. 1C  shows results using primers and a probe designed to be specific for L536_D538&gt;P that shows good specificity detecting mutant compared to wild type sample, with wild type sample showing no signal. 
         FIG. 2A ,  FIG. 2B , and  FIG. 2C  also compare discrimination of primers and probes for detecting the L536R mutant and wild type samples. The amplification growth curves show cycles on the X axis and signal on the Y axis.  FIG. 2A  shows results using the L536R_RS01 allele-specific primer that shows poor discrimination between wild type and mutant.  FIG. 2B  shows results using the L536R_RS09 allele-specific primer that shows better discrimination between wild type and mutant.  FIG. 2C  shows results using the L536R_RS04 allele-specific primer that shows good specificity detecting mutant compared to wild type sample, with wild type sample showing essentially no signal. 
         FIG. 3  shows specificity of primers and probe designed to be specific for the P535H mutation compared to other ESR1 mutant samples. 
         FIG. 4A  shows amplification growth curves for an E380Q mutation positive control (MC, top/left two curves), a E380Q mutation positive sample (IN008, next lower two curves), E380 wild type (FFPET_WT, next lower two curves), and non-template control (NTC, flat bottom line). 
         FIG. 4B  shows the same amplification growth curves without the E380Q mutation positive control curves. 
         FIG. 5  shows amplification growth curves for a D538G mutation positive control (MC, top/left curve), 3 D538G mutation positive samples (D538G, next lower three curves), D538 wild type (next lower 48 curves), and non-template control (NTC, flat bottom line). 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     I. INTRODUCTION 
     Provided herein is a multiplex PCR assay for detecting the presence of mutations in the human estrogen receptor (ESR1). These mutations are associated with resistance to hormone therapy in cancer patients with hormone responsive cancers (ESR1 positive and/or progesterone receptor positive) that were sensitive to hormone therapy when initially administered. The assays can be carried out with non-invasive samples (e.g., blood, plasma, serum, urine, etc.) so that repeated testing can be carried out in a patient on hormone therapy without taking multiple tissue biopsies. 
     The presently described assays rely on proven, widely adopted technology and provide accurate, reproducible, and rapid results. 
     II. DEFINITIONS 
     The terms “estrogen receptor”, “ER”, and “ESR1” are used interchangeably herein unless otherwise noted. ESR1 can also be used to refer to the gene encoding the ER protein. 
     The term “multiplex” refers to an assay in which more than one target is detected. The terms “receptacle,” “vessel,” “tube,” “well,” “chamber,” “microchamber,” etc. refer to a container that can hold reagents or an assay. If the receptacle is in a kit and holds reagents, or is being used for an amplification reaction, it can be closed or sealed to avoid contamination or evaporation. If the receptacle is being used for an assay, it can be open or accessible, at least during set up of the assay. 
     The terms “individually detected” or “individual detection,” referring to a marker gene or marker gene product, indicates that each marker in a multiplex reaction is detected. That is, each marker is associated with a different label (detected by a differently labeled probe). 
     The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” refer to polymers of nudeotides (e.g., ribonucleotides or deoxyribo-nudeotides) and includes naturally-occurring (adenosine, guanidine, cytosine, uracil and thymidine), non-naturally occurring, and modified nucleic acids. The term is not limited by length (e.g., number of monomers) of the polymer. A nucleic acid may be single-stranded or double-stranded and will generally contain 5′-3′ phosphodiester bonds, although in some cases, nucleotide analogs may have other linkages. Monomers are typically referred to as nucleotides. The term “non-natural nucleotide” or “modified nucleotide” refers to a nucleotide that contains a modified nitrogenous base, sugar or phosphate group, or that incorporates a non-natural moiety in its structure. Examples of non-natural nucleotides include dideoxynucleotides, biotinylated, aminated, deaminated, alkylated, benzylated and fluorophor-labeled nudeotides. 
     The term “primer” refers to a short nucleic acid (an oligonucleotide) that acts as a point of initiation of polynucleotide strand synthesis by a nucleic acid polymerase under suitable conditions. Polynucleotide synthesis and amplification reactions typically include an appropriate buffer, dNTPs and/or rNTPs, and one or more optional cofactors, and are carried out at a suitable temperature. A primer typically includes at least one target-hybridized region that is at least substantially complementary to the target sequence (e.g., having 0, 1, 2, or 3 mismatches). This region of is typically about 8 to about 40 nucleotides in length, e.g., 12-25 nucleotides. A “primer pair” refers to a forward and reverse primer that are oriented in opposite directions relative to the target sequence, and that produce an amplification product in amplification conditions. In some embodiments, multiple primer pairs rely on a single common forward or reverse primer. For example, multiple allele-specific forward primers can be considered part of a primer pair with the same, common reverse primer, e.g., if the multiple alleles are in close proximity to each other. 
     As used herein, “probe” means any molecule that is capable of selectively binding to a specifically intended target biomolecule, for example, a nucleic acid sequence of interest that hybridizes to the probes. The probe is detectably labeled with at least one non-nucleotide moiety. In some embodiments, the probe is labeled with a fluorophore and quencher. 
     The words “complementary” or “complementarity” refer to the ability of a nucleic acid in a polynucleotide to form a base pair with another nucleic acid in a second polynucleotide. For example, the sequence A-G-T (A-G-U for RNA) is complementary to the sequence T-C-A (U-C-A for RNA). Complementarity may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing. A probe or primer is considered “specific for” a target sequence if it is at least partially complementary to the target sequence. Depending on the conditions, the degree of complementarity to the target sequence is typically higher for a shorter nucleic acid such as a primer (e.g., greater than 80%, 90%, 95%, or higher) than for a longer sequence. In some embodiments, the term “each primer pair specific for a different sequence in the ESR1 gene” indicates that each primer pair specifically amplifies a different sequence, e.g., a different allele or mutation, of the ESR1 gene. 
     The term “specifically amplifies” indicates that a primer set amplifies a target sequence more than non-target sequence at a statistically significant level. The term “specifically detects” indicates that a probe will detect a target sequence more than non-target sequence at a statistically significant level. As will be understood in the art, specific amplification and detection can be determined using a negative control, e.g., a sample that includes the same nucleic acids as the test sample, but not the target sequence or a sample lacking nucleic acids. For example, primers and probes that specifically amplify and detect a target sequence result in a Ct that is readily distinguishable from background (non-target sequence), e.g., a Ct that is at least 2, 3, 4, 5, 5-10, 10-20, or 10-30 cycles less than background. The term “allele-specific” PCR refers to amplification of a target sequence using primers that specifically amplify a particular allelic variant of the target sequence. Typically, the forward or reverse primer includes the exact complement of the allelic variant at that position. 
     The terms “identical” or “percent identity,” in the context of two or more nucleic acids, or two or more polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides, or amino acids, that are the same (e.g., about 60% identity, e.g., at least any of 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters, or by manual alignment and visual inspection. See e.g., the NCBI web site at ncbi.nlm.nih.gov/BLAST. Such sequences are then said to be “substantially identical.” Percent identity is typically determined over optimally aligned sequences, so that the definition applies to sequences that have deletions and/or additions, as well as those that have substitutions. The algorithms commonly used in the art account for gaps and the like. Typically, identity exists over a region comprising an a sequence that is at least about 8-25 amino acids or nucleotides in length, or over a region that is 50-100 amino acids or nucleotides in length, or over the entire length of the reference sequence. 
     The term “kit” refers to any manufacture (e.g., a package or a container) including at least one reagent, such as a nucleic acid probe or probe pool or the like, for specifically amplifying, capturing, tagging/converting or detecting RNA or DNA as described herein. 
     The term “amplification conditions” refers to conditions in a nucleic acid amplification reaction (e.g., PCR amplification) that allow for hybridization and template-dependent extension of the primers. The term “amplicon” or “amplification product” refers to a nucleic acid molecule that contains all or a fragment of the target nucleic acid sequence and that is formed as the product of in vitro amplification by any suitable amplification method. One of skill will understand that a forward and reverse primer (primer pair) defines the borders of an amplification product. The term “generate an amplification product” when applied to primers, indicates that the primers, under appropriate conditions (e.g., in the presence of a nucleotide polymerase and NTPs), will produce the defined amplification product. Various PCR conditions are described in PCR Strategies (Innis et al., 1995, Academic Press, San Diego, Calif.) at Chapter 14;  PCR Protocols: A Guide to Methods and Applications  (Innis et al., Academic Press, NY, 1990) 
     The term “amplification product” refers to the product of an amplification reaction. The amplification product includes the primers used to initiate each round of polynucleotide synthesis. An “amplicon” is the sequence targeted for amplification, and the term can also be used to refer to amplification product. The 5′ and 3′ borders of the amplicon are defined by the forward and reverse primers. 
     The terms “individual”, “subject”, and “patient” are used interchangeably herein. The individual can be pre-diagnosis, post-diagnosis but pre-therapy, undergoing therapy, or post-therapy. In the context of the present disclosure, the individual is typically seeking medical care. 
     The term “sample” or “biological sample” refers to any composition containing or presumed to contain nucleic acid. The term includes purified or separated components of cells, tissues, or blood, e.g., DNA, RNA, proteins, cell-free portions, or cell lysates. The sample can be FFPET, e.g., from a tumor or metastatic lesion. The sample can also be from frozen or fresh tissue, or from a liquid sample, e.g., blood or a blood component (plasma or serum), urine, semen, saliva, sputum, mucus, semen, tear, lymph, cerebral spinal fluid, mouth/throat rinse, bronchial alveolar lavage, material washed from a swab, etc. Samples also may include constituents and components of in vitro cultures of cells obtained from an individual, including cell lines. The sample can also be partially processed from a sample directly obtained from an individual, e.g., cell lysate or blood depleted of red blood cells. 
     The term “obtaining a sample from an individual” means that a biological sample from the individual is provided for testing. The obtaining can be directly from the individual, or from a third party that directly obtained the sample from the individual. 
     The term “providing therapy for an individual” means that the therapy is prescribed, recommended, or made available to the individual. The therapy may be actually administered to the individual by a third party (e.g., an in-patient injection), or by the individual herself. 
     A “control” sample or value refers to a value that serves as a reference, usually a known reference, for comparison to a test sample or test conditions. For example, a test sample can be taken from a test condition, e.g., from an individual suspected of having cancer, and compared to samples from known conditions, e.g., from a cancer-free individual (negative control), or from an individual known to have cancer (positive control). In the context of the present disclosure, the test sample is typically from a breast cancer patient. A control can also represent an average value or a range gathered from a number of tests or results. A control can also be prepared for reaction conditions. For example, a control for the presence, quality, and/ or quantity of nucleic acid (e.g., internal control) can include primers or probes that will detect a sequence known to be present in the sample (e.g., a housekeeping gene such as beta actin, beta globin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein L37 and L38, PPlase, EIF3, eukaryotic translation elongation factor 2 (eEF2), DHFR, or succinate dehydrogenase). In some embodiments, the internal control can be a sequence from a region of the same gene that is not commonly variant (e.g., in a different exon). A known added polynucleotide, e.g., having a designated length, can also be added. An example of a negative control is one free of nucleic acids, or one including primers or probes specific for a sequence that would not be present in the sample, e.g., from a different species. One of skill will understand that the selection of controls will depend on the particular assay, e.g., so that the control is cell type and organism-appropriate. One of skill in the art will recognize that controls can be designed for assessment of any number of parameters. For example, a control can be devised to compare therapeutic benefit based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of benefit and/or side effects). Controls can be designed for in vitro applications. One of skill in the art will understand which controls are valuable in a given situation and be able to analyze data based on comparisons to control values. Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant. 
     The terms “label,” “tag,” “detectable moiety,” and like terms refer to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include fluorescent dyes (fluorophores), luminescent agents, radioisotopes (e.g.,  32 P,  3 H), electron-dense reagents, or an affinity-based moiety, e.g., a poly-A (interacts with poly-T) or poly-T tag (interacts with poly-A), a His tag (interacts with Ni), or a strepavidin tag (separable with biotin). One of skill will understand that a detectable label conjugated to a nucleic acid is not naturally occurring. 
     Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Lackie, DICTIONARY OF CELL AND MOLECULAR BIOLOGY, Elsevier (4th ed. 2007); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, N.Y. 1989). The term “a” or “an” is intended to mean “one or more.” The terms “comprise,” “comprises,” and “comprising,” when preceding the recitation of a step or an element, are intended to mean that the addition of further steps or elements is optional and not excluded. 
     III. NUCLEIC ACID SAMPLES 
     Samples for nucleic acid amplification can be obtained from any source suspected of containing nucleic acid. Samples can be taken from formalin fixed paraffin embedded tissue (FFPET), tissue biopsy, or cultured cells (e.g., obtained from a patient, or representing a control). In some embodiments, the sample is obtained in a non-invasive manner, e.g., from urine, skin, swab, saliva, blood or a blood fraction. 
     In a sample that includes cells, the cells can be separated out (e.g., using size-based filtration or centrifugation), thereby leaving cell free nucleic acids (cfNA), including nucleic acids in exosomes, microvesicles, viral particles, or those circulating freely. Alternatively, the cells can be lysed to obtain cellular nucleic acids, either in the presence of magnetic glass particles (MGPs) or before addition of the cellular lysate to the MGPs. 
     Methods for isolating nucleic acids from biological samples are known, e.g., as described in Sambrook, and several kits are commercially available (e.g., High Pure RNA Isolation Kit, High Pure Viral Nudeic Acid Kit, and MagNA Pure LC Total Nudeic Acid Isolation Kit, DNA Isolation Kit for Cells and Tissues, DNA Isolation Kit for Mammalian Blood, High Pure FFPET DNA Isolation Kit, available from Roche). In the context of the presently disclosed methods, RNA is collected, though in some embodiments, the classifier can be used on previously prepared cDNA. 
     IV. ESTROGEN RECEPTOR MUTATED CANCER AND THERAPIES 
     Hormone sensitive tumors are commonly treated with hormone therapy. Hormone insensitive tumors typically do not respond to hormone therapy. The term “hormone therapy” applies to a variety of treatments that block the effect of hormones that affect growth of the tumor. In some cases however, such as certain womb and kidney cancers, progesterone or a synthetic version thereof is prescribed. 
     Breast cancer is often hormone sensitive, and patients diagnosed with breast cancer are tested to determine if the tumor is estrogen receptor (ESR1) and/ or progesterone receptor positive. Receptor positive tumors can be treated with agents that interfere with production of these hormones. Ovarian ablation can be carried out to remove ovaries using surgery or radiation. Surgery is often followed up with additional chemotherapy. Selective estrogen receptor modulators (SERMS) that block the effect of estrogen on the ESR include tamoxifen, raloxifene, toremifene, and fulvestrant. Additional treatments include aromatase inhibitors (e.g., anastrozole, exemestane, letrozole) and lutenizing hormone (LH) blockers (e.g., goserelin, leuprolide). These therapies can be used in combination, e.g., a SERM with an aromatase inhibitor for post-menopausal patient, or a SERM with an LH blocker for pre-menopausal patient. Similar therapies are considered effective for receptor positive ovarian cancers, while prostate cancer can be treated with lutenizing hormone blockers, anti-androgens, and/ or gonadotrophin releasing hormone blockers. 
     In addition, patients can benefit from standard chemotherapy. This can include CHOP (cyclophosphamide; doxorubicin; vincristine; and prednisolone) or R-CHOP, which further includes rituximab and/or etoposide. The cocktail can be administered periodically for a set period of time, or until reduction in tumor size and/or symptoms are detected. For example, the CHOP or R-CHOP can be administered every 2 or 3 weeks. 
     Regardless of which treatment is selected, it typically begins with a low dose so that side effects can be determined, and the dose increased, e.g., until side effects appear or within the patient&#39;s tolerance, or until clinical benefit is observed. 
     After initial treatment, patients can become resistant to hormone therapy. Resistance to hormone therapy is thought to be due, at least in part, to mutations in the estrogen receptor. Many of these mutations are in the ligand binding domain, so that the receptor is active in the absence of estrogen release. Examples include K303R, E380Q, V392I, S463P, K531E, V534E, P535H, L536Q/R, Y537S/C/N, D538G, and R555C. Testing for mutations in the estrogen receptor allows for more effective therapeutic decisions for patients. The testing can be periodic during hormone therapy, e.g., before, during, or after observation of resistance. For example, a patient receiving SERM therapy can be switched to a different hormone therapy or combination thereof, or to standard chemotherapy. 
     V. AMPLIFICATION AND DETECTION 
     A nucleic acid sample can be used for detection and quantification, e.g., using nucleic acid amplification, e.g., using any primer-dependent method. In some embodiments, a preliminary reverse transcription step is carried out (also referred to as RT-PCR, not to be confused with real time PCR). See, e.g., Hierro et al. (2006) 72:7148. The term “qRT-PCR” as used herein refers to reverse transcription and quantitative PCR. Both reactions can be carried out in a single tube without interruption, e.g., to add reagents. For example, a polyT primer can be used to reverse transcribe all mRNAs in a sample with a polyA tail, random oligonucleotides can be used, or a primer can be designed that is specific for a particular target transcript that will be reverse transcribed into cDNA. The cDNA, or DNA from the sample, can form the initial template to be for quantitative amplification (real time or quantitative PCR, i.e., RTPCR or qPCR). qPCR allows for reliable detection and measurement of products generated during each cycle of PCR process. Such techniques are well known in the art, and kits and reagents are commercially available, e.g., from Roche Molecular Systems, Life Technologies, Bio-Rad, etc. See, e.g., Pfaffl (2010)  Methods: The ongoing evolution of qPCR  vol. 50. 
     A separate reverse transcriptase and thermostable DNA polymerase can be used, e.g., in a two-step (reverse transcription followed by addition of DNA polymerase and amplification) or combined reaction (with both enzymes added at once). In some embodiments, the target nucleic acid is amplified with a thermostable polymerase with both reverse transcriptase activity and DNA template-dependent activity. Exemplary enzymes include Tth DNA polymerase, the C. therm Polymerase system, and those disclosed in 0520140170730 and US20140051126. 
     Probes for use as described herein can be labeled with a fluorophore and quencher (e.g., TaqMan, LightCycler, Molecular Beacon, Scorpion, or Dual Labeled probes). Appropriate fluorophores include FAM, JOE, TET, Cal Fluor Gold 540, HEX, VIC, Cal Fluor Orang 560, TAMRA, Cyanine 3, Quasar 570, Cal Fluor Red 590, Rox, Texas Red, Cyanine 5, Quasar 670, and Cyanine 5.5. Appropriate quenchers include TAMRA (for FAM, JOE, and TET), DABCYL, and BHQ1-3. 
     Detection devices are known in the art and can be selected as appropriate for the selected labels. Detection devices appropriate for quantitative PCR include the cobas® and Light Cycler® systems (Roche), PRISM 7000 and 7300 real-time PCR systems (Applied Biosystems), etc. Six-channel detection is available on the CFX96 Real Time PCR Detection System (Bio-Rad) and Rotorgene Q (Qiagen), allowing for a higher degree of multiplexing. 
     Results can be expressed in terms of a threshold cycle (abbreviated as Ct, and in some instances Cq or Cp). A lower Ct value reflects the rapid achievement of a predetermined threshold level, e.g., because of higher target nucleic acid concentration or a more efficient amplification. A higher Ct value may reflect lower target nucleic acid concentration, or inefficient or inhibited amplification. The threshold cycle is generally selected to be in the linear range of amplification for a given target. In some embodiments, the Ct is set as the cycle at which the growth signal exceeds a pre-defined threshold line, e.g., in relation to the baseline, or by determining the maximum of the second derivation of the growth curve. Determination of Ct is known in the art, and described, e.g., in U.S. Pat. No. 7363168. 
     VI. KITS 
     Provided herein are kits for carrying out multiplex, allele-specific PCR to detect ESR1 mutations. The kits include primers and probes for specifically detecting particular ESR1 mutations as described herein. 
     A primer pair and probe for detecting ESR1 V422DelV can be selected from forward primer sequences SEQ ID NOs:284 or 287-300, reverse primer sequences SEQ ID NOs:285 or 302-311, and probe sequences SEQ ID NOs:286 or 301. A primer pair and probe for detecting ESR1 S463P can be selected from forward primer sequences SEQ ID NOs:260 or 263-272, reverse primer sequences SEQ ID NOs:261 or 274-283, and probe sequences SEQ ID NOs:262 or 273. A primer pair and probe for detecting ESR1 L536H can be selected from forward primer sequences SEQ ID NOs:96-105, reverse primer sequences SEQ ID NOs:106-120, and probe sequences SEQ ID NOs:94 or 95. A primer pair for detecting ESR1 L536P can be selected from forward primer sequences SEQ ID NOs:121-130 and reverse primer sequences SEQ ID NOs:131-140. A primer pair for detecting ESR1 L536Q can be selected from forward primer sequences SEQ ID NOs:141-150 and reverse primer sequences SEQ ID NOs:151-160. A primer pair for detecting ESR1 L536R can be selected from forward primer sequences SEQ ID NOs:161-170 and reverse primer sequences SEQ ID NOs:171-180. A primer pair for detecting ESR1 D538G can be selected from forward primer sequences SEQ ID NOs:4-13 and reverse primer sequences SEQ ID NOs:14-27 or 550. A primer pair and probe for detecting ESR1 K303R can be selected from forward primer sequences SEQ ID NOs:473, 474, or 478-487, reverse primer sequences SEQ ID NOs:477, 489-498 or 576, and probe sequences SEQ ID NOs:477 or 488. A primer pair and probe for detecting ESR1 E380Q can be selected from forward primer sequences SEQ ID NOs:28 or 32-42, reverse primer sequences SEQ ID NOs:29, 30, or 44-53, and probe sequences SEQ ID NOs:43 or 54. Primers for detecting ESR1 L536_D538&gt;P can be selected from reverse primer sequences SEQ ID NOs:84-93. A primer pair and probe for detecting ESR1 Y537C can be selected from forward primer sequences SEQ ID NOs:394 or 397-406, reverse primer sequences SEQ ID NOs:395, 408-425, or 574, and probe sequences SEQ ID NOs:396 or 407. A primer pair for detecting ESR1 Y537N can be selected from forward primer sequences SEQ ID NOs:426-435 and reverse primer sequences SEQ ID NOs:436-448 or 575-577. A primer pair for detecting ESR1 Y537S can be selected from forward primer sequences SEQ ID NOs:449-458 and reverse primer sequences SEQ ID NOs:459-472 or 578. A primer pair and probe for detecting ESR1 S341L can be selected from forward primer sequences SEQ ID NOs:232, 233, or 238-247, reverse primer sequences SEQ ID NOs:234, 235, or 250-259, and probe sequences SEQ ID NOs:248 or 249. A primer pair and probe for detecting ESR1 L429V can be selected from forward primer sequences SEQ ID NOs:58 or 61-70, reverse primer sequences SEQ ID NOs:59 or 72-81, and probe sequences SEQ ID NOs:60 or 71. A primer pair and probe for detecting ESR1 V533M can be selected from forward primer sequences SEQ ID NOs:312-318, 322-333, 355, or 356, reverse primer sequences SEQ ID NOs:319, 320, or 336354, and probe sequences SEQ ID NOs:321, 334, or 335. A primer pair and probe for detecting ESR1 V534E can be selected from forward primer sequences SEQ ID NOs:357 o r363-373, reverse primer sequences SEQ ID NOs:358-362 or 378-393, and probe sequences SEQ ID NOs:363 or 374-377. A primer pair for detecting ESR1 P535H can be selected from forward primer sequences SEQ ID NOs:181-190 and reverse primer sequences SEQ ID NOs:191-231. 
     In addition, for mutations in exon 8, a common forward or common reverse primer can be used to amplify each amplification product, and a common probe can be used to detect each amplification product. One of skill will understand that slight variations can be made to the sequences, e.g., addition or deletion of 1-3 nucleotides. 
     Allele-specific primers selected from those disclosed in Table 5 for each mutation can be included in the kit, along with common primers and probes corresponding to that mutation. 
     In some embodiments, the kit comprises 1-18 stock solutions comprising allele-specific primer pairs and probes. The stock solutions can optionally include DNA polymerase, and optionally include a primer pair and probe to detect an internal control. In some embodiment, the kit includes 2-5 such stock solutions, e.g., 2, 3, 4, or 5 stock solutions. 
     In some embodiments, the stock solution mixtures further comprise buffers, dNTPs, and other elements (e.g., cofactors or aptamers) appropriate for reverse transcription and amplification. Typically, the mixture is concentrated, so that an aliquot is added to the final reaction volume, along with sample (e.g., DNA), enzymes, and/ or water. In some embodiments, the kit further comprises reverse transcriptase (or an enzyme with reverse transcriptase activity), and/or DNA polymerase (e.g., thermostable DNA polymerase such as Taq, ZO5, and derivatives thereof). 
     In some embodiments, the kit further includes components for DNA or RNA purification from a sample, e.g., a non-invasive or tissue sample. For example, the kit can include components from MagNA Pure LC Total Nucleic Acid Isolation Kit, DNA Isolation Kit for Cells and Tissues, DNA Isolation Kit for Mammalian Blood, High Pure FFPET DNA Isolation Kit, High Pure or MagNA Pure RNA Isolation Kits (Roche), DNeasy or RNeasy Kits (Qiagen), PureLink DNA or RNA Isolation Kits (Thermo Fisher), etc. 
     In some embodiments, the kit further includes at least one control sample, e.g., nucleic acids from non-cancer sample (or pooled samples), or from a known ESR1 mutated sample (or pooled samples). In some embodiments, the kit includes a negative control, e.g., lacking nucleic acids, or lacking mutated ESR1 nucleic acids. In some embodiments, the kit further includes consumables, e.g., plates or tubes for nucleic acid preparation, tubes for sample collection, etc. In some embodiments, the kit further includes instructions for use, reference to a website, or software. 
     VII. EXAMPLES 
     Assay Design 
     Multiplex, allele-specific PCR assays to detect 18 mutations in the ESR1 gene were designed. The mutations detected are shown in Table 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 ESR1 mutations 
               
            
           
           
               
               
               
               
            
               
                   
                 ESR1 mutation (AA) 
                 ESR1 mutation (NA) 
                 Exon 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 K303R 
                 908 A &gt; G 
                 4 
               
               
                 2 
                 S341L 
                 1022 C &gt; T 
                 4 
               
               
                 3 
                 E380Q 
                 1138 G &gt; C 
                 5 
               
               
                 4 
                 V422DelV 
                 1262_1264delTGG 
                 6 
               
               
                 5 
                 L429V 
                 1285 C &gt; G 
                 6 
               
               
                 6 
                 S463P 
                 1387 T &gt; C 
                 7 
               
               
                 7 
                 V533M 
                 1597 G &gt; A 
                 8 
               
               
                 8 
                 V534E 
                 1601 T &gt; A 
                 8 
               
               
                 9 
                 P535H 
                 1604 C &gt; A 
                 8 
               
               
                 10 
                 L536Q 
                 1607/8 TC &gt; AC 
                 8 
               
               
                 11 
                 L536R 
                 1607 T &gt; G 
                 8 
               
               
                 12 
                 L536H 
                 1607 T &gt; A 
                 8 
               
               
                 13 
                 L536P 
                 1607 T &gt; C 
                 8 
               
               
                 14 
                 L536_D538 &gt; P 
                 1607_1614 TCTATGAC &gt; CC 
                 8 
               
               
                 15 
                 Y537N 
                 1609 T &gt; A 
                 8 
               
               
                 16 
                 Y537C 
                 1610 A &gt; G 
                 8 
               
               
                 17 
                 Y537S 
                 1610 A &gt; C 
                 8 
               
               
                 18 
                 D538G 
                 1613 A &gt; G 
                 8 
               
               
                   
               
            
           
         
       
     
     The primers and probes generated for the assays are shown in Table 2. One of skill will understand that the list is not exhaustive, and that primers and/or probe sequences can be slightly modified from those shown, e.g., adding or subtracting one or a few nucleotides, shifting the position of the modified nudeotide, or using a different modified nudeotide at a given site (e.g., N4_Et_dC instead of t_BB_dC). The mutation detected is indicated in the primer/ probe name. FS# and RS# refer to forward specific primer and reverse specific primer, respectively. CF# and CR# refer to common forward primer and common reverse primer, respectively. F_P# and R_P# refer to forward and reverse probes. While probes in exon 8 are labeled for specific mutations, they can be used to detect any exon 8 mutation; the allele specificity for this assay is determined by allele-specific primers. As an example, for detecting the D538G mutation, any ESRLD538G_FS# primer can be used with any ESRL[Exon8mut]_CR# primer and any ESRL[Exon8mut]_F_P# probe, and any ESRLD538G_RS# primer can be used with any ESRL[Exon8mut]_CF# primer and any ESRL[Exon8mut]_R_P# probe. 
     Modifications include t_BB_[dNTP]=N6-tert-butyl-benzyl [dNTP]; N4_Et_[dNTP]=N4-Ethyl [dNTP]; N4_Bz_[dNTP]=N4-benzyl [dNTP]; N6_Me_[dNTP]=N6-methyl [dNTP]; pdU=5-propenyl-deoxyuracil; 7_Dz_dG=7-deaza dG; LNA-[dNTP]=locked nucleic acid [dNTP]. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Primer and probe sequences 
               
            
           
           
               
               
               
               
            
               
                 SEQ 
                   
                   
                   
               
               
                 ID NO 
                 OLIGO NAME 
                 SEQUENCE 
                 MODIFICATION 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 ESR1_EXWT1_FS03 
                 TGGCCCTACTGCATCAGATCCAA 
                   
               
               
                   
               
               
                 2 
                 ESR1_EXWT1_FS04 
                 ATCAGATCCAAGGGAACGAGCT 
                   
               
               
                   
               
               
                 3 
                 ESR1_EXWT1_RS03 
                 GCTTGCTGCTGTCCAGGTACA 
                   
               
               
                   
               
               
                 4 
                 ESR1_D538G_FS01 
                 CGTGGTGCCCCTCTATGG 
                   
               
               
                   
               
               
                 5 
                 ESR1_D538G_FS02 
                 CGTGGTGCCCCTCTATAG 
                   
               
               
                   
               
               
                 6 
                 ESR1_D538G_FS03 
                 CGTGGTGCCCCTCTATCG 
                   
               
               
                   
               
               
                 7 
                 ESR1_D538G_FS04 
                 CGTGGTGCCCCTCTATTG 
                   
               
               
                   
               
               
                 8 
                 ESR1_D538G_FS05 
                 CGTGGTGCCCCTCTAAGG 
                   
               
               
                   
               
               
                 9 
                 ESR1_D538G_FS06 
                 CGTGGTGCCCCTCTACGG 
                   
               
               
                   
               
               
                 10 
                 ESR1_D538G_FS07 
                 CGTGGTGCCCCTCTAGGG 
                   
               
               
                   
               
               
                 11 
                 ESR1_D538G_FS08 
                 CGTGGTGCCCCTCTCTGG 
                   
               
               
                   
               
               
                 12 
                 ESR1_D538G_FS09 
                 CGTGGTGCCCCTCTGTGG 
                   
               
               
                   
               
               
                 13 
                 ESR1_D538G_FS10 
                 CGTGGTGCCCCTCTTTGG 
                   
               
               
                   
               
               
                 14 
                 ESR1_D538G_RS01 
                 GCATCTCCAGCAGC1GGC 
                 1 = A or optional 
               
               
                   
                   
                   
                 t_BB_dA 
               
               
                   
               
               
                 15 
                 ESR1_D538G_RS02 
                 GCATCTCCAGCAGCAGAC 
                   
               
               
                   
               
               
                 16 
                 ESR1_D538G_RS03 
                 GCATCTCCAGCAGCAGCC 
                   
               
               
                   
               
               
                 17 
                 ESR1_D538G_RS04 
                 GCATCTCCAGCAGCAGTC 
                   
               
               
                   
               
               
                 18 
                 ESR1_D538G_RS05 
                 GCATCTCCAGCAGCAAGC 
                   
               
               
                   
               
               
                 19 
                 ESR1_D538G_RS06 
                 GCATCTCCAGCAGCACGC 
                   
               
               
                   
               
               
                 20 
                 ESR1_D538G_RS07 
                 GCATCTCCAGCAGCATGC 
                   
               
               
                   
               
               
                 21 
                 ESR1_D538G_RS08 
                 GCATCTCCAGCAG1CGGC 
                 1 = C or optional 
               
               
                   
                   
                   
                 N4_Et_dC or 
               
               
                   
                   
                   
                 t_BB_dC 
               
               
                   
               
               
                 22 
                 ESR1_D538G_RS09 
                 GCATCTCCAGCAG1GGGC 
                 1 = C or optional 
               
               
                   
                   
                   
                 N4_Et_dC or 
               
               
                   
                   
                   
                 t_BB_dC 
               
               
                   
               
               
                 23 
                 ESR1_D538G_RS10 
                 GCATCTCCAGCAG1TGGC 
                 1 = C or optional 
               
               
                   
                   
                   
                 N4_Et_dC or 
               
               
                   
                   
                   
                 t_BB_dC 
               
               
                   
               
               
                 24 
                 ESR1_D538G_RS11 
                 GCATCTCCAGCAGCG1GC 
                 1 = G or optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 25 
                 ESR1_D538G_RS12 
                 CAGCATCTCCAGCAGCTGGC 
                   
               
               
                   
               
               
                 26 
                 ESR1_D538G_TBBDC_RS01 
                 CAGCATCTCCAGCAG1AGGC 
                 1 = C or optional 
               
               
                   
                   
                   
                 t_BB_dC 
               
               
                   
               
               
                 27 
                 ESR1_D538G_TBBDC_RS09B 
                 CAGCATCTCCAGCAG1GGGC 
                 1 = C or optional 
               
               
                   
                   
                   
                 t_BB_dC 
               
               
                   
               
               
                 28 
                 ESR1_E380Q_CF01 
                 TGAGTCAGCAGGGTTT 
                   
               
               
                   
               
               
                 29 
                 ESR1_E380Q_CR01 
                 TAGGAGCAAACAGTAGCTTCC 
                   
               
               
                   
               
               
                 30 
                 ESR1_E380Q_CR02 
                 AGTTAGGAGCAAACAGTAGCTTCC 
                   
               
               
                   
               
               
                 31 
                 ESR1_E380Q_F_P01 
                 1TGTGCCT2GGCTAGAGATCCTGATGATTGGT3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 32 
                 ESR1_E380Q_FS01 
                 CCATGATCAGGTCCACCTT1TAC 
                 1 = C or optional 
               
               
                   
                   
                   
                 t_BB_dC 
               
               
                   
               
               
                 33 
                 ESR1_E380Q_FS02 
                 CCATGATCAGGTCCACCTTCTCC 
                   
               
               
                   
               
               
                 34 
                 ESR1_E380Q_FS03 
                 CCATGATCAGGTCCACCTTCTGC 
                   
               
               
                   
               
               
                 35 
                 ESR1_E380Q_FS04 
                 CCATGATCAGGTCCACCTTCTTC 
                   
               
               
                   
               
               
                 36 
                 ESR1_E380Q_FS05 
                 CCATGATCAGGTCCACCTTCAAC 
                   
               
               
                   
               
               
                 37 
                 ESR1_E380Q_FS06 
                 CCATGATCAGGTCCACCTTCCAC 
                   
               
               
                   
               
               
                 38 
                 ESR1_E380Q_FS07 
                 CCATGATCAGGTCCACCTTCGAC 
                   
               
               
                   
               
               
                 39 
                 ESR1_E380Q_FS08 
                 CCATGATCAGGTCCACCTTATAC 
                   
               
               
                   
               
               
                 40 
                 ESR1_E380Q_FS09 
                 CCATGATCAGGTCCACCTTGTAC 
                   
               
               
                   
               
               
                 41 
                 ESR1_E380Q_FS10 
                 CCATGATCAGGTCCACCTTTTAC 
                   
               
               
                   
               
               
                 42 
                 ESR1_E380Q_FS11 
                 CTCCATGATCAGGTCCACCTTGTAC 
                   
               
               
                   
               
               
                 43 
                 ESR1_E380Q_R_P01 
                 1TGGACCTG2ATCATGGAGGGTCAAATCCACA3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 44 
                 ESR1_E380Q_RS01 
                 GGATCTCTAGCCAGGCACATTG 
                   
               
               
                   
               
               
                 45 
                 ESR1_E380Q_RS02 
                 GGATCTCTAGCCAGGCACATAG 
                   
               
               
                   
               
               
                 46 
                 ESR1_E380Q_RS03 
                 GGATCTCTAGCCAGGCACATCG 
                   
               
               
                   
               
               
                 47 
                 ESR1_E380Q_RS04 
                 GGATCTCTAGCCAGGCACATGG 
                   
               
               
                   
               
               
                 48 
                 ESR1_E380Q_RS05 
                 GGATCTCTAGCCAGGCACAATG 
                   
               
               
                   
               
               
                 49 
                 ESR1_E380Q_RS06 
                 GGATCTCTAGCCAGGCACACTG 
                   
               
               
                   
               
               
                 50 
                 ESR1_E380Q_RS07 
                 GGATCTCTAGCCAGGCACAGTG 
                   
               
               
                   
               
               
                 51 
                 ESR1_E380Q_RS08 
                 GGATCTCTAGCCAGGCACCTTG 
                   
               
               
                   
               
               
                 52 
                 ESR1_E380Q_RS09 
                 GGATCTCTAGCCAGGCACGTTG 
                   
               
               
                   
               
               
                 53 
                 ESR1_E380Q_RS10 
                 GGATCTCTAGCCAGGCACTTTG 
                   
               
               
                   
               
               
                 54 
                 ESR1_EXWT1_F_P01 
                 1CCCTGAAC2CGTCCGCAGCTCAAGAT3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 55 
                 ESR1_EXWT1_FS01 
                 CCCTACTGCATCAGATCCAA 
                   
               
               
                   
               
               
                 56 
                 ESR1_EXWT1_FS02 
                 ATCAGATCCAAGGGAACGAG 
                   
               
               
                   
               
               
                 57 
                 ESR1_EXWT1_RS01 
                 TTGCTGCTGTCCAGGTACA 
                   
               
               
                   
               
               
                 58 
                 ESR1_L429V_CF01 
                 TGCTATGTTTTCATAGGAACCAGG 
                   
               
               
                   
               
               
                 59 
                 ESR1_L429V_CR01 
                 GATTTGAGGCACACAAACTCCT 
                   
               
               
                   
               
               
                 60 
                 ESR1_L429V_F_P01 
                 1TCATCTCGGT2TCCGCATGATGAATCTGCAG3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 61 
                 ESR1_L429V_FS01 
                 TGGAGATCTTCGACATG1TGG 
                 1 = C or optional 
               
               
                   
                   
                   
                 N4_Bz_dC or 
               
               
                   
                   
                   
                 N4_Et_dC or 
               
               
                   
                   
                   
                 t_BB_dC 
               
               
                   
               
               
                 62 
                 ESR1_L429V_FS02 
                 TGGAGATCTTCGACATGCTAG 
                   
               
               
                   
               
               
                 63 
                 ESR1_L429V_FS03 
                 TGGAGATCTTCGACATGCTCG 
                   
               
               
                   
               
               
                 64 
                 ESR1_L429V_FS04 
                 TGGAGATCTTCGACATGCTTG 
                   
               
               
                   
               
               
                 65 
                 ESR1_L429V_FS05 
                 TGGAGATCTTCGACATGCAGG 
                   
               
               
                   
               
               
                 66 
                 ESR1_L429V_FS06 
                 TGGAGATCTTCGACATGCCGG 
                   
               
               
                   
               
               
                 67 
                 ESR1_L429V_FS07 
                 TGGAGATCTTCGACATGCGGG 
                   
               
               
                   
               
               
                 68 
                 ESR1_L429V_FS08 
                 TGGAGATCTTCGACATGATGG 
                   
               
               
                   
               
               
                 69 
                 ESR1_L429V_FS09 
                 TGGAGATCTTCGACATGGTGG 
                   
               
               
                   
               
               
                 70 
                 ESR1_L429V_FS10 
                 TGGAGATCTTCGACATGTTGG 
                   
               
               
                   
               
               
                 71 
                 ESR1_L429V_R_P01 
                 1TCGAAGATCTC2CACCATGCCCTCTACACAT3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 72 
                 ESR1_L429V_RS01 
                 GGAACCGAGATGATGTAGCCAC 
                   
               
               
                   
               
               
                 73 
                 ESR1_L429V_RS02 
                 GGAACCGAGATGATGTAGCCCC 
                   
               
               
                   
               
               
                 74 
                 ESR1_L429V_RS03 
                 GGAACCGAGATGATGTAGCCGC 
                   
               
               
                   
               
               
                 75 
                 ESR1_L429V_RS04 
                 GGAACCGAGATGATGTAGCCTC 
                   
               
               
                   
               
               
                 76 
                 ESR1_L429V_RS05 
                 GGAACCGAGATGATGTAGCAAC 
                   
               
               
                   
               
               
                 77 
                 ESR1_L429V_RS06 
                 GGAACCGAGATGATGTAGCGAC 
                   
               
               
                   
               
               
                 78 
                 ESR1_L429V_RS07 
                 GGAACCGAGATGATGTAGCTAC 
                   
               
               
                   
               
               
                 79 
                 ESR1_L429V_RS08 
                 GGAACCGAGATGATGTAGACAC 
                   
               
               
                   
               
               
                 80 
                 ESR1_L429V_RS09 
                 GGAACCGAGATGATGTAGGCAC 
                   
               
               
                   
               
               
                 81 
                 ESR1_L429V_RS10 
                 GGAACCGAGATGATGTAGTCAC 
                   
               
               
                   
               
               
                 82 
                 ESR1_L536_CF01 
                 CTTTCTGTGTCTTCCCACCTACAG 
                   
               
               
                   
               
               
                 83 
                 ESR1_L536_CR01 
                 AAGTGGCTTTGGTCCGTC 
                   
               
               
                   
               
               
                 84 
                 ESR1_L536_D538&gt;P_RS01 
                 CCAGCAGCAGG1GGG 
                 1 = G or optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 85 
                 ESR1_L536_D538&gt;P_RS02 
                 CAGCAGCAGGG1GG 
                 1 = G or optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 86 
                 ESR1_L536_D538&gt;P_RS03 
                 CCAGCAGCAGG1GG 
                 1 = G or optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 87 
                 ESR1_L536_D538&gt;P_RS04 
                 TCCAGCAGCAGG1GG 
                 1 = G or optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 88 
                 ESR1_L536_D538&gt;P_RS05 
                 CTCCAGCAGCAGG1GG 
                 1 = G or optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 89 
                 ESR1_L536_D538&gt;P_RS06 
                 CTCCAGCAGCAGG1G 
                 1 = G or optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 90 
                 ESR1_L536_D538&gt;P_RS07 
                 TCTCCAGCAGCAGG1G 
                 1 = G or optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 91 
                 ESR1_L536_D538&gt;P_RS08 
                 ATCTCCAGCAGCAGG1G 
                 1 = G or optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 92 
                 ESR1_L536_D538&gt;P_RS09 
                 CATCTCCAGCAGCAGGG 
                   
               
               
                   
               
               
                   
               
               
                 93 
                 ESR1_L536_D538&gt;P_RS10 
                 GCATCTCCAGCAGCAGGG 
                   
               
               
                   
               
               
                 94 
                 ESR1_L536_F_P01 
                 1TACATG2CGCCCACTAGCCGTGGA3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 95 
                 ESR1_L536_R_P01 
                 1TGCACTT2CATGCTGTACAGATGCTCCATGC3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 96 
                 ESR1_L536H_FS01 
                 GCAAGAACGTGGTGCCCCA 
                   
               
               
                   
               
               
                 97 
                 ESR1_L536H_FS02 
                 GCAAGAACGTGGTGCCCAA 
                   
               
               
                   
               
               
                 98 
                 ESR1_L536H_FS03 
                 GCAAGAACGTGGTGCCCGA 
                   
               
               
                   
               
               
                 99 
                 ESR1_L536H_FS04 
                 GCAAGAACGTGGTGCCCTA 
                   
               
               
                   
               
               
                 100 
                 ESR1_L536H_FS05 
                 GCAAGAACGTGGTGCCACA 
                   
               
               
                   
               
               
                 101 
                 ESR1_L536H_FS06 
                 GCAAGAACGTGGTGCCGCA 
                   
               
               
                   
               
               
                 102 
                 ESR1_L536H_FS07 
                 GCAAGAACGTGGTGCCTCA 
                   
               
               
                   
               
               
                 103 
                 ESR1_L536H_FS08 
                 GCAAGAACGTGGTGCACCA 
                   
               
               
                   
               
               
                 104 
                 ESR1_L536H_FS09 
                 GCAAGAACGTGGTGCGCCA 
                   
               
               
                   
               
               
                 105 
                 ESR1_L536H_FS10 
                 GCAAGAACGTGGTGCTCCA 
                   
               
               
                   
               
               
                 106 
                 ESR1_L536H_RS01 
                 CTCCAGCAGCAGGTCAT1GT 
                 1 = A or optional 
               
               
                   
                   
                   
                 t_BB_dA 
               
               
                   
               
               
                 107 
                 ESR1_L536H_RS02 
                 CTCCAGCAGCAGGTCATAAT 
                   
               
               
                   
               
               
                 108 
                 ESR1_L536H_RS03 
                 CTCCAGCAGCAGGTCATACT 
                   
               
               
                   
               
               
                 109 
                 ESR1_L536H_RS04 
                 CTCCAGCAGCAGGTCATATT 
                   
               
               
                   
               
               
                 110 
                 ESR1_L536H_RS05 
                 CTCCAGCAGCAGGTCATCGT 
                   
               
               
                   
               
               
                 111 
                 ESR1_L536H_RS06 
                 CTCCAGCAGCAGGTCATGGT 
                   
               
               
                   
               
               
                 112 
                 ESR1_L536H_RS07 
                 CTCCAGCAGCAGGTCATTGT 
                   
               
               
                   
               
               
                 113 
                 ESR1_L536H_RS08 
                 CTCCAGCAGCAGGTCAAAGT 
                   
               
               
                   
               
               
                 114 
                 ESR1_L536H_RS09 
                 CTCCAGCAGCAGGTCACAGT 
                   
               
               
                   
               
               
                 115 
                 ESR1_L536H_RS10 
                 CTCCAGCAGCAGGTCAGAGT 
                   
               
               
                   
               
               
                 116 
                 ESR1_L536H_RS11 
                 CATCTCCAGCAGCAGGTCATAAT 
                   
               
               
                   
               
               
                 117 
                 ESR1_L536H_RS12 
                 CATCTCCAGCAGCAGGTCATATT 
                   
               
               
                   
               
               
                 118 
                 ESR1_L536H_RS13 
                 CATCTCCAGCAGCAGGTCGTAGT 
                   
               
               
                   
               
               
                 119 
                 ESR1_L536H_RS14 
                 CATCTCCAGCAGCAGGTCTTAGT 
                   
               
               
                   
               
               
                 120 
                 ESR1_L536H_RS15 
                 CATCTCCAGCAGCAGGTCCTAGT 
                   
               
               
                   
               
               
                 121 
                 ESR1_L536P_FS01 
                 AGAACGTGGTGCCCCC 
                   
               
               
                   
               
               
                 122 
                 ESR1_L536P_FS02 
                 AGAACGTGGTGCCCAC 
                   
               
               
                   
               
               
                 123 
                 ESR1_L536P_FS03 
                 AGAACGTGGTGCCCGC 
                   
               
               
                   
               
               
                 124 
                 ESR1_L536P_FS04 
                 AGAACGTGGTGCCCTC 
                   
               
               
                   
               
               
                 125 
                 ESR1_L536P_FS05 
                 AGAACGTGGTGCCACC 
                   
               
               
                   
               
               
                 126 
                 ESR1_L536P_FS06 
                 AGAACGTGGTGCCGCC 
                   
               
               
                   
               
               
                 127 
                 ESR1_L536P_FS07 
                 AGAACGTGGTGCCTCC 
                   
               
               
                   
               
               
                 128 
                 ESR1_L536P_FS08 
                 AGAACGTGGTGCACCC 
                   
               
               
                   
               
               
                 129 
                 ESR1_L536P_FS09 
                 AGAACGTGGTGCGCCC 
                   
               
               
                   
               
               
                 130 
                 ESR1_L536P_FS10 
                 AGAACGTGGTGCTCCC 
                   
               
               
                   
               
               
                 131 
                 ESR1_L536P_RS01 
                 CTCCAGCAGCAGGTCAT1GG 
                 1 = A or optional 
               
               
                   
                   
                   
                 t_BB_dA 
               
               
                   
               
               
                 132 
                 ESR1_L536P_RS02 
                 CTCCAGCAGCAGGTCATAAG 
                   
               
               
                   
               
               
                 133 
                 ESR1_L536P_RS03 
                 CTCCAGCAGCAGGTCATACG 
                   
               
               
                   
               
               
                 134 
                 ESR1_L536P_RS04 
                 CTCCAGCAGCAGGTCATATG 
                   
               
               
                   
               
               
                 135 
                 ESR1_L536P_RS05 
                 CTCCAGCAGCAGGTCATCGG 
                   
               
               
                   
               
               
                 136 
                 ESR1_L536P_RS06 
                 CTCCAGCAGCAGGTCATGGG 
                   
               
               
                   
               
               
                 137 
                 ESR1_L536P_RS07 
                 CTCCAGCAGCAGGTCATTGG 
                   
               
               
                   
               
               
                 138 
                 ESR1_L536P_RS08 
                 CTCCAGCAGCAGGTCAAAGG 
                   
               
               
                   
               
               
                 139 
                 ESR1_L536P_RS09 
                 CTCCAGCAGCAGGTCACAGG 
                   
               
               
                   
               
               
                 140 
                 ESR1_L536P_RS10 
                 CTCCAGCAGCAGGTCAGAGG 
                   
               
               
                   
               
               
                 141 
                 ESR1_L536Q_FS01 
                 AGAACGTGGTGCCCCAG 
                   
               
               
                   
               
               
                 142 
                 ESR1_L536Q_FS02 
                 AGAACGTGGTGCCCCCG 
                   
               
               
                   
               
               
                 143 
                 ESR1_L536Q_FS03 
                 AGAACGTGGTGCCCCGG 
                   
               
               
                   
               
               
                 144 
                 ESR1_L536Q_FS04 
                 AGAACGTGGTGCCCCTG 
                   
               
               
                   
               
               
                 145 
                 ESR1_L536Q_FS05 
                 AGAACGTGGTGCCCAAG 
                   
               
               
                   
               
               
                 146 
                 ESR1_L536Q_FS06 
                 AGAACGTGGTGCCCGAG 
                   
               
               
                   
               
               
                 147 
                 ESR1_L536Q_FS07 
                 AGAACGTGGTGCCCTAG 
                   
               
               
                   
               
               
                 148 
                 ESR1_L536Q_FS08 
                 AGAACGTGGTGCCACAG 
                   
               
               
                   
               
               
                 149 
                 ESR1_L536Q_FS09 
                 AGAACGTGGTGCCGCAG 
                   
               
               
                   
               
               
                 150 
                 ESR1_L536Q_FS10 
                 AGAACGTGGTGCCTCAG 
                   
               
               
                   
               
               
                 151 
                 ESR1_L536Q_RS01 
                 CTCCAGCAGCAGGTCAT1CT 
                 1 = A or optional 
               
               
                   
                   
                   
                 t_BB_dA 
               
               
                   
               
               
                 152 
                 ESR1_L536Q_RS02 
                 CTCCAGCAGCAGGTCATAAT 
                   
               
               
                   
               
               
                 153 
                 ESR1_L536Q_RS03 
                 CTCCAGCAGCAGGTCATAGT 
                   
               
               
                   
               
               
                 154 
                 ESR1_L536Q_RS04 
                 CTCCAGCAGCAGGTCATATT 
                   
               
               
                   
               
               
                 155 
                 ESR1_L536Q_RS05 
                 CTCCAGCAGCAGGTCATCCT 
                   
               
               
                   
               
               
                 156 
                 ESR1_L536Q_RS06 
                 CTCCAGCAGCAGGTCATGCT 
                   
               
               
                   
               
               
                 157 
                 ESR1_L536Q_RS07 
                 CTCCAGCAGCAGGTCATTCT 
                   
               
               
                   
               
               
                 158 
                 ESR1_L536Q_RS08 
                 CTCCAGCAGCAGGTCAAACT 
                   
               
               
                   
               
               
                 159 
                 ESR1_L536Q_RS09 
                 CTCCAGCAGCAGGTCACACT 
                   
               
               
                   
               
               
                 160 
                 ESR1_L536Q_RS10 
                 CTCCAGCAGCAGGTCAGACT 
                   
               
               
                   
               
               
                 161 
                 ESR1_L536R_FS01 
                 AGAACGTGGTGCCCCG 
                   
               
               
                   
               
               
                 162 
                 ESR1_L536R_FS02 
                 AGAACGTGGTGCCCAG 
                   
               
               
                   
               
               
                 163 
                 ESR1_L536R_FS03 
                 AGAACGTGGTGCCCGG 
                   
               
               
                   
               
               
                 164 
                 ESR1_L536R_FS04 
                 AGAACGTGGTGCCCTG 
                   
               
               
                   
               
               
                 165 
                 ESR1_L536R_FS05 
                 AGAACGTGGTGCCACG 
                   
               
               
                   
               
               
                 166 
                 ESR1_L536R_FS06 
                 AGAACGTGGTGCCGCG 
                   
               
               
                   
               
               
                 167 
                 ESR1_L536R_FS07 
                 AGAACGTGGTGCCTCG 
                   
               
               
                   
               
               
                 168 
                 ESR1_L536R_FS08 
                 AGAACGTGGTGCACCG 
                   
               
               
                   
               
               
                 169 
                 ESR1_L536R_FS09 
                 AGAACGTGGTGCGCCG 
                   
               
               
                   
               
               
                 170 
                 ESR1_L536R_FS10 
                 AGAACGTGGTGCTCCG 
                   
               
               
                   
               
               
                 171 
                 ESR1_L536R_RS01 
                 CTCCAGCAGCAGGTCAT1GC 
                 1 = A or optional 
               
               
                   
                   
                   
                 t_BB_dA 
               
               
                   
               
               
                 172 
                 ESR1_L536R_RS02 
                 CTCCAGCAGCAGGTCATAAC 
                   
               
               
                   
               
               
                 173 
                 ESR1_L536R_RS03 
                 CTCCAGCAGCAGGTCATACC 
                   
               
               
                   
               
               
                 174 
                 ESR1_L536R_RS04 
                 CTCCAGCAGCAGGTCATATC 
                   
               
               
                   
               
               
                 175 
                 ESR1_L536R_RS05 
                 CTCCAGCAGCAGGTCATCGC 
                   
               
               
                   
               
               
                 176 
                 ESR1_L536R_RS06 
                 CTCCAGCAGCAGGTCATGGC 
                   
               
               
                   
               
               
                 177 
                 ESR1_L536R_RS07 
                 CTCCAGCAGCAGGTCATTGC 
                   
               
               
                   
               
               
                 178 
                 ESR1_L536R_RS08 
                 CTCCAGCAGCAGGTCAAAGC 
                   
               
               
                   
               
               
                 179 
                 ESR1_L536R_RS09 
                 CTCCAGCAGCAGGTCACAGC 
                   
               
               
                   
               
               
                 180 
                 ESR1_L536R_RS10 
                 CTCCAGCAGCAGGTCAGAGC 
                   
               
               
                   
               
               
                 181 
                 ESR1_P535H_FS01 
                 AAGTGCAAGAACGTGGTGC1 
                 1 = A or optional 
               
               
                   
                   
                   
                 N6_Bz_dA 
               
               
                   
               
               
                 182 
                 ESR1_P535H_FS02 
                 AAGTGCAAGAACGTGGTGAA 
                   
               
               
                   
               
               
                 183 
                 ESR1_P535H_FS03 
                 AAGTGCAAGAACGTGGTGGA 
                   
               
               
                   
               
               
                 184 
                 ESR1_P535H_FS04 
                 AAGTGCAAGAACGTGGTGTA 
                   
               
               
                   
               
               
                 185 
                 ESR1_P535H_FS05 
                 AAGTGCAAGAACGTGGTACA 
                   
               
               
                   
               
               
                 186 
                 ESR1_P535H_FS06 
                 AAGTGCAAGAACGTGGTCCA 
                   
               
               
                   
               
               
                 187 
                 ESR1_P535H_FS07 
                 AAGTGCAAGAACGTGGTTCA 
                   
               
               
                   
               
               
                 188 
                 ESR1_P535H_FS08 
                 AAGTGCAAGAACGTGGAGCA 
                   
               
               
                   
               
               
                 189 
                 ESR1_P535H_FS09 
                 AAGTGCAAGAACGTGGCGCA 
                   
               
               
                   
               
               
                 190 
                 ESR1_P535H_FS10 
                 AAGTGCAAGAACGTGGGGCA 
                   
               
               
                   
               
               
                 191 
                 ESR1_P535H_RS01 
                 AGCAGCAGGTCATAGAGGT 
                   
               
               
                   
               
               
                 192 
                 ESR1_P535H_RS02 
                 AGCAGCAGGTCATAGAGAT 
                   
               
               
                   
               
               
                 193 
                 ESR1_P535H_RS03 
                 AGCAGCAGGTCATAGAGCT 
                   
               
               
                   
               
               
                 194 
                 ESR1_P535H_RS04 
                 AGCAGCAGGTCATAGAGTT 
                   
               
               
                   
               
               
                 195 
                 ESR1_P535H_RS05 
                 AGCAGCAGGTCATAGAAGT 
                   
               
               
                   
               
               
                 196 
                 ESR1_P535H_RS06 
                 AGCAGCAGGTCATAGACGT 
                   
               
               
                   
               
               
                 197 
                 ESR1_P535H_RS07 
                 AGCAGCAGGTCATAGATGT 
                   
               
               
                   
               
               
                 198 
                 ESR1_P535H_RS08 
                 AGCAGCAGGTCATAGCGGT 
                   
               
               
                   
               
               
                 199 
                 ESR1_P535H_RS09 
                 AGCAGCAGGTCATAGGGGT 
                   
               
               
                   
               
               
                 200 
                 ESR1_P535H_RS10 
                 AGCAGCAGGTCATAGTGGT 
                   
               
               
                   
               
               
                 201 
                 ESR1_P535H_RS11 
                 CCAGCAGCAGGTCAT1GAGGT 
                 1 = A or optional 
               
               
                   
                   
                   
                 t_BB_dA 
               
               
                   
               
               
                 202 
                 ESR1_P535H_RS12 
                 CCAGCAGCAGGTCATG1GGT 
                 1 = A or C or 
               
               
                   
                   
                   
                 optional t_BB_dA 
               
               
                   
                   
                   
                 or t_BB_dC 
               
               
                   
               
               
                 203 
                 ESR1_P535H_RS13 
                 CCAGCAGCAGGTCATAGGGT 
                   
               
               
                   
               
               
                 204 
                 ESR1_P535H_RS14 
                 CCAGCAGCAGGTCAT1GTGGT 
                 1 = A or optional 
               
               
                   
                   
                   
                 t_BB_dA 
               
               
                   
               
               
                 205 
                 ESR1_P535H_RS15 
                 AGCAGCAGGTCATAAAGGT 
                   
               
               
                   
               
               
                 206 
                 ESR1_P535H_RS16 
                 AGCAGCAGGTCATACAGGT 
                   
               
               
                   
               
               
                 207 
                 ESR1_P535H_RS17 
                 AGCAGCAGGTCATATAGGT 
                   
               
               
                   
               
               
                 208 
                 ESR1_P535H_RS18 
                 AGCAGCAGGTCATAGAGG1 
                 1 = Optional LNA-T 
               
               
                   
               
               
                 209 
                 ESR1_P535H_RS19 
                 AGCAGCAGGTCATAGAG1T 
                 1 = Optional LNA-G 
               
               
                   
               
               
                 210 
                 ESR1_P535H_RS20 
                 AGCAGCAGGTCATAGA1GT 
                 1 = Optional LNA-G 
               
               
                   
               
               
                 211 
                 ESR1_P535H_RS21 
                 AGCAGCAGGTCATAG1GGT 
                 1 = Optional LNA-A 
               
               
                   
               
               
                 212 
                 ESR1_P535H_RS22 
                 CCAGCAGCAGGTCATAG1GGT 
                 1 = C or optional 
               
               
                   
                   
                   
                 N4_Et_dC 
               
               
                   
               
               
                 213 
                 ESR1_P535H_RS23 
                 AGCAGCAGGTCATAAACGT 
                   
               
               
                   
               
               
                 214 
                 ESR1_P535H_RS24 
                 AGCAGCAGGTCATACACGT 
                   
               
               
                   
               
               
                 215 
                 ESR1_P535H_RS25 
                 AGCAGCAGGTCATATACGT 
                   
               
               
                   
               
               
                 216 
                 ESR1_P535H_RS26 
                 AGCAGCAGGTCATAAATGT 
                   
               
               
                   
               
               
                 217 
                 ESR1_P535H_RS27 
                 AGCAGCAGGTCATACATGT 
                   
               
               
                   
               
               
                 218 
                 ESR1_P535H_RS28 
                 AGCAGCAGGTCATATATGT 
                   
               
               
                   
               
               
                 219 
                 ESR1_P535H_RS29 
                 AGCAGCAGGTCATAAAAGT 
                   
               
               
                   
               
               
                 220 
                 ESR1_P535H_RS30 
                 AGCAGCAGGTCATACAAGT 
                   
               
               
                   
               
               
                 221 
                 ESR1_P535H_RS31 
                 AGCAGCAGGTCATATAAGT 
                   
               
               
                   
               
               
                 222 
                 ESR1_P535H_RS32 
                 AGCAGCAGGTCAT1GAGGT 
                 1 = A or optional 
               
               
                   
                   
                   
                 N6_Me_dA 
               
               
                   
               
               
                 223 
                 ESR1_P535H_RS33 
                 CCAGCAGCAGGTCATAG1GGT 
                 1 = A or optional 
               
               
                   
                   
                   
                 N6_Me_dA 
               
               
                   
               
               
                 224 
                 ESR1_P535H_RS34 
                 CCAGCAGCAGGTCAT1GTGGT 
                 1 = A or optional 
               
               
                   
                   
                   
                 N6_Me_dA 
               
               
                   
               
               
                 225 
                 ESR1_P535H_RS35 
                 AGCAGCAGGTCATAG1GGT 
                 1 = A or optional 
               
               
                   
                   
                   
                 t_BB_dA 
               
               
                   
               
               
                 226 
                 ESR1_P535H_RS36 
                 CAGCAGCAGGTCATAAAGGT 
                   
               
               
                   
               
               
                 227 
                 ESR1_P535H_RS37 
                 CAGCAGCAGGTCATACAGGT 
                   
               
               
                   
               
               
                 228 
                 ESR1_P535H_RS38 
                 CAGCAGCAGGTCATATAGGT 
                   
               
               
                   
               
               
                 229 
                 ESR1_P535H_RS39 
                 CCAGCAGCAGGTCATAAAGGT 
                   
               
               
                   
               
               
                 230 
                 ESR1_P535H_RS40 
                 CCAGCAGCAGGTCATACAGGT 
                   
               
               
                   
               
               
                 231 
                 ESR1_P535H_RS41 
                 CCAGCAGCAGGTCATATAGGT 
                   
               
               
                   
               
               
                 232 
                 ESR1_S341L_CF01 
                 AGATGGTCAGTGCCTTGTTG 
                   
               
               
                   
               
               
                 233 
                 ESR1_S341L_CF02 
                 CAGATGGTCAGTGCCTTGTT 
                   
               
               
                   
               
               
                 234 
                 ESR1_S341L_CR01 
                 ATTCTTACCTGGCACCCTCTTC 
                   
               
               
                   
               
               
                 235 
                 ESR1_S341L_CR02 
                 CTCTTCGCCCAGTTGATCAT 
                   
               
               
                   
               
               
                 236 
                 ESR1_S341L_F_P01 
                 1AGACAGG2GAGCTGGTTCACATGATCAACTG3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 237 
                 ESR1_S341L_F_P02 
                 1TGATGG2GCTTACTGACCAACCTGGCAGACA3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 238 
                 ESR1_S341L_FS01 
                 CCAGACCCTTCAGTGAAG1TTT 
                 1 = C or optional 
               
               
                   
                   
                   
                 t_BB_dC 
               
               
                   
               
               
                 239 
                 ESR1_S341L_FS02 
                 CCAGACCCTTCAGTGAAGCTAT 
                   
               
               
                   
               
               
                 240 
                 ESR1_S341L_FS03 
                 CCAGACCCTTCAGTGAAGCTCT 
                   
               
               
                   
               
               
                 241 
                 ESR1_S341L_FS04 
                 CCAGACCCTTCAGTGAAGCTGT 
                   
               
               
                   
               
               
                 242 
                 ESR1_S341L_FS05 
                 CCAGACCCTTCAGTGAAGCATT 
                   
               
               
                   
               
               
                 243 
                 ESR1_S341L_FS06 
                 CCAGACCCTTCAGTGAAGCCTT 
                   
               
               
                   
               
               
                 244 
                 ESR1_S341L_FS07 
                 CCAGACCCTTCAGTGAAGCGTT 
                   
               
               
                   
               
               
                 245 
                 ESR1_S341L_FS08 
                 CCAGACCCTTCAGTGAAGATTT 
                   
               
               
                   
               
               
                 246 
                 ESR1_S341L_FS09 
                 CCAGACCCTTCAGTGAAGGTTT 
                   
               
               
                   
               
               
                 247 
                 ESR1_S341L_FS10 
                 CCAGACCCTTCAGTGAAGTTTT 
                   
               
               
                   
               
               
                 248 
                 ESR1_S341L_R_P01 
                 1ACTGAAG2GGTCTGGTAGGATCATACTCGGA3 
                   
               
               
                   
               
               
                 249 
                 ESR1_S341L_R_P02 
                 1CTGAAG23GTCTGGTAGGATCATACTCGGAATA4 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = G or optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
                   
                   
                 4 = Phosphate 
               
               
                   
               
               
                 250 
                 ESR1_S341L_RS01 
                 GTTGGTCAGTAAGCCCATCATCA 
                   
               
               
                   
               
               
                 251 
                 ESR1_S341L_RS02 
                 GTTGGTCAGTAAGCCCATCATAA 
                   
               
               
                   
               
               
                 252 
                 ESR1_S341L_RS03 
                 GTTGGTCAGTAAGCCCATCATGA 
                   
               
               
                   
               
               
                 253 
                 ESR1_S341L_RS04 
                 GTTGGTCAGTAAGCCCATCATTA 
                   
               
               
                   
               
               
                 254 
                 ESR1_S341L_RS05 
                 GTTGGTCAGTAAGCCCATCAACA 
                   
               
               
                   
               
               
                 255 
                 ESR1_S341L_RS06 
                 GTTGGTCAGTAAGCCCATCACCA 
                   
               
               
                   
               
               
                 256 
                 ESR1_S341L_RS07 
                 GTTGGTCAGTAAGCCCATCAGCA 
                   
               
               
                   
               
               
                 257 
                 ESR1_S341L_RS08 
                 GTTGGTCAGTAAGCCCATCCTCA 
                   
               
               
                   
               
               
                 258 
                 ESR1_S341L_RS09 
                 GTTGGTCAGTAAGCCCATCGTCA 
                   
               
               
                   
               
               
                 259 
                 ESR1_S341L_RS10 
                 GTTGGTCAGTAAGCCCATCTTCA 
                   
               
               
                   
               
               
                 260 
                 ESR1_S463P_CF01 
                 CTAGACCTCATCCTCTTTGAGC 
                   
               
               
                   
               
               
                 261 
                 ESR1_S463P_CR01 
                 CCATCAGGTGGATCAAAGTGTCTG 
                   
               
               
                   
               
               
                 262 
                 ESR1_S463P_F_P01 
                 1ACCATAT2CCACCGAGTCCTGGACAAGATCA3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 263 
                 ESR1_S463P_FS01 
                 CATTCAGGAGTGTACACATTT1TGC 
                 1 = C or optional 
               
               
                   
                   
                   
                 t_BB_dC 
               
               
                   
               
               
                 264 
                 ESR1_S463P_FS02 
                 CATTCAGGAGTGTACACATTTCTAC 
                   
               
               
                   
               
               
                 265 
                 ESR1_S463P_FS03 
                 CATTCAGGAGTGTACACATTTCTCC 
                   
               
               
                   
               
               
                 266 
                 ESR1_S463P_FS04 
                 CATTCAGGAGTGTACACATTTCTTC 
                   
               
               
                   
               
               
                 267 
                 ESR1_S463P_FS05 
                 CATTCAGGAGTGTACACATTTCAGC 
                   
               
               
                   
               
               
                 268 
                 ESR1_S463P_FS06 
                 CATTCAGGAGTGTACACATTTCCGC 
                   
               
               
                   
               
               
                 269 
                 ESR1_S463P_FS07 
                 CATTCAGGAGTGTACACATTTCGGC 
                   
               
               
                   
               
               
                 270 
                 ESR1_S463P_FS08 
                 CATTCAGGAGTGTACACATTTATGC 
                   
               
               
                   
               
               
                 271 
                 ESR1_S463P_FS09 
                 CATTCAGGAGTGTACACATTTGTGC 
                   
               
               
                   
               
               
                 272 
                 ESR1_S463P_FS10 
                 CATTCAGGAGTGTACACATTTTTGC 
                   
               
               
                   
               
               
                 273 
                 ESR1_S463P_R_P01 
                 1TGTGTAC2ACTCCTGAATGCGCAGAGAGAGA3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 274 
                 ESR1_S463P_RS01 
                 AGACTTCAGGGTGCTGGG 
                   
               
               
                   
               
               
                 275 
                 ESR1_S463P_RS11 
                 CAGACTTCAGGGTGCTGAG 
                   
               
               
                   
               
               
                 276 
                 ESR1_S463P_RS03 
                 AGACTTCAGGGTGCTGCG 
                   
               
               
                   
               
               
                 277 
                 ESR1_S463P_RS04 
                 AGACTTCAGGGTGCTGTG 
                   
               
               
                   
               
               
                 278 
                 ESR1_S463P_RS05 
                 AGACTTCAGGGTGCTAGG 
                   
               
               
                   
               
               
                 279 
                 ESR1_S463P_RS06 
                 AGACTTCAGGGTGCTCGG 
                   
               
               
                   
               
               
                 280 
                 ESR1_S463P_RS07 
                 AGACTTCAGGGTGCTTGG 
                   
               
               
                   
               
               
                 281 
                 ESR1_S463P_RS08 
                 AGACTTCAGGGTGCAGGG 
                   
               
               
                   
               
               
                 282 
                 ESR1_S463P_RS09 
                 AGACTTCAGGGTGCCGGG 
                   
               
               
                   
               
               
                 283 
                 ESR1_S463P_RS10 
                 AGACTTCAGGGTGCGGGG 
                   
               
               
                   
               
               
                 284 
                 ESR1_V422DELV_CF01 
                 GTCTTGTGGAAGATTTTCTGT 
                   
               
               
                   
               
               
                 285 
                 ESR1_V422DELV_CR01 
                 TTGAGGCACACAAACTCCTC 
                   
               
               
                   
               
               
                 286 
                 ESR1_V422DELV_F_P01 
                 1TGGCTACATCA2TCTCGGTTCCGCATGATGA3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 287 
                 ESR1_V422DELV_FS01 
                 ATGTGTAGAGGGCATGG1GAT 
                 1 = A or optional 
               
               
                   
                   
                   
                 t_BB_dA 
               
               
                   
               
               
                 288 
                 ESR1_V422DELV_FS02 
                 ATGTGTAGAGGGCATGGAGCT 
                   
               
               
                   
               
               
                 289 
                 ESR1_V422DELV_FS03 
                 ATGTGTAGAGGGCATGGAGGT 
                   
               
               
                   
               
               
                 290 
                 ESR1_V422DELV_FS04 
                 ATGTGTAGAGGGCATGGAGTT 
                   
               
               
                   
               
               
                 291 
                 ESR1_V422DELV_FS05 
                 ATGTGTAGAGGGCATGGAAAT 
                   
               
               
                   
               
               
                 292 
                 ESR1_V422DELV_FS06 
                 ATGTGTAGAGGGCATGGACAT 
                   
               
               
                   
               
               
                 293 
                 ESR1_V422DELV_FS07 
                 ATGTGTAGAGGGCATGGATAT 
                   
               
               
                   
               
               
                 294 
                 ESR1_V422DELV_FS08 
                 ATGTGTAGAGGGCATGGCGAT 
                   
               
               
                   
               
               
                 295 
                 ESR1_V422DELV_FS09 
                 ATGTGTAGAGGGCATG12GAT 
                 1 = G or optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
                   
                   
                 2 = G or optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 296 
                 ESR1_V422DELV_FS10 
                 ATGTGTAGAGGGCATGGTGAT 
                   
               
               
                   
               
               
                 297 
                 ESR1_V422DELV_FS14 
                 AAATGTGTAGAG1GCATGGCGAT 
                 1 = G or optional 
               
               
                   
                   
                   
                 d_I 
               
               
                   
               
               
                 298 
                 ESR1_V422DELV_FS15 
                 AAATGTGTAGAGGGCATGGTGAT 
                   
               
               
                   
               
               
                 299 
                 ESR1_V422DELV_FS16 
                 AAATGTGTAGAGGGCATGTAGAT 
                   
               
               
                   
               
               
                 300 
                 ESR1_V422DELV_FS17 
                 AAATGTGTAGAGGGCATGAAGAT 
                   
               
               
                   
               
               
                 301 
                 ESR1_V422DELV_R_P01 
                 1CCTCTACACATT2TTCCCTGGTTCCTATGA3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 302 
                 ESR1_V422DELV_RS01 
                 GCAGCATGTCGAAGATCTCCAT 
                   
               
               
                   
               
               
                 303 
                 ESR1_V422DELV_RS02 
                 GCAGCATGTCGAAGATCTCCCT 
                   
               
               
                   
               
               
                 304 
                 ESR1_V422DELV_RS03 
                 GCAGCATGTCGAAGATCTCCGT 
                   
               
               
                   
               
               
                 305 
                 ESR1_V422DELV_RS04 
                 GCAGCATGTCGAAGATCTCCTT 
                   
               
               
                   
               
               
                 306 
                 ESR1_V422DELV_RS05 
                 GCAGCATGTCGAAGATCTCAAT 
                   
               
               
                   
               
               
                 307 
                 ESR1_V422DELV_RS06 
                 GCAGCATGTCGAAGATCTCGAT 
                   
               
               
                   
               
               
                 308 
                 ESR1_V422DELV_RS07 
                 GCAGCATGTCGAAGATCTCTAT 
                   
               
               
                   
               
               
                 309 
                 ESR1_V422DELV_RS08 
                 GCAGCATGTCGAAGATCTACAT 
                   
               
               
                   
               
               
                 310 
                 ESR1_V422DELV_RS09 
                 GCAGCATGTCGAAGATCTGCAT 
                   
               
               
                   
               
               
                 311 
                 ESR1_V422DELV_RS10 
                 GCAGCATGTCGAAGATCTTCAT 
                   
               
               
                   
               
               
                 312 
                 ESR1_V533M_CF01 
                 GTAGTCCTTTCTGTGTCTTCCC 
                   
               
               
                   
               
               
                 313 
                 ESR1_V533M_CF02 
                 CTTTCTGTGTCTTCCCACCTAC 
                   
               
               
                   
               
               
                 314 
                 ESR1_V533M_CF03 
                 TGTCTTCCCACCTACAGTAACAAA 
                   
               
               
                   
               
               
                 315 
                 ESR1_V533M_CF04 
                 CTCTAAAGTAGTCCTTTCTGTGTCTTC 
                   
               
               
                   
               
               
                 316 
                 ESR1_V533M_CF05 
                 TCTAAAGTAGTCCTTTCTGTGTCTTC 
                   
               
               
                   
               
               
                 317 
                 ESR1_V533M_CF06 
                 TAAAGTAGTCCTTTCTGTGTCTTCC 
                   
               
               
                   
               
               
                 318 
                 ESR1_V533M_CF07 
                 AGTAGTCCTTTCTGTGTCTTCC 
                   
               
               
                   
               
               
                 319 
                 ESR1_V533M_CR01 
                 GCTAGTGGGCGCATGTA 
                   
               
               
                   
               
               
                 320 
                 ESR1_V533M_CR02 
                 CTAGTGGGCGCATGTA 
                   
               
               
                   
               
               
                 321 
                 ESR1_V533M_F_P01 
                 1TCTATG2ACCTGCTGCTGGAGATGCTGGA3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 322 
                 ESR1_V533M_FS01 
                 ACAGCATGAAGTGCAAGAA12 
                 1 = C or optional 
               
               
                   
                   
                   
                 N4_Bz_dC or 
               
               
                   
                   
                   
                 N4_Et_dC 
               
               
                   
                   
                   
                 2 = A or optional 
               
               
                   
                   
                   
                 N6_Bz_dA 
               
               
                   
               
               
                 323 
                 ESR1_V533M_FS02 
                 ACAGCATGAAGTGCAAGAAAA 
                   
               
               
                   
               
               
                 324 
                 ESR1_V533M_FS03 
                 ACAGCATGAAGTGCAAGAAGA 
                   
               
               
                   
               
               
                 325 
                 ESR1_V533M_FS04 
                 ACAGCATGAAGTGCAAGAATA 
                   
               
               
                   
               
               
                 326 
                 ESR1_V533M_FS05 
                 ACAGCATGAAGTGCAAGACCA 
                   
               
               
                   
               
               
                 327 
                 ESR1_V533M_FS06 
                 ACAGCATGAAGTGCAAGAGCA 
                   
               
               
                   
               
               
                 328 
                 ESR1_V533M_FS07 
                 ACAGCATGAAGTGCAAGATCA 
                   
               
               
                   
               
               
                 329 
                 ESR1_V533M_FS08 
                 ACAGCATGAAGTGCAAGCACA 
                   
               
               
                   
               
               
                 330 
                 ESR1_V533M_FS09 
                 ACAGCATGAAGTGCAAGGACA 
                   
               
               
                   
               
               
                 331 
                 ESR1_V533M_FS10 
                 ACAGCATGAAGTGCAAGTACA 
                   
               
               
                   
               
               
                 332 
                 ESR1_V533M_FS12 
                 TGTACAGCATGAAGTGCAAGCACA 
                   
               
               
                   
               
               
                 333 
                 ESR1_V533M_FS13 
                 TGTACAGCATGAAGTGCAAGGACA 
                   
               
               
                   
               
               
                 334 
                 ESR1_V533M_R_P01 
                 1TGCACT2TCATGCTGTACAGATGCTCCATGC3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 335 
                 ESR1_V533M_R_P02 
                 1TGCACTT2CATGCTGTACAGATGCTCCATGC3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 336 
                 ESR1_V533M_RS01 
                 GGTCATAGAG1GGCACCAT 
                 1 = Optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 337 
                 ESR1_V533M_RS018 
                 GGTCATAGAG1GGCA2CAT 
                 1 = Optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
                   
                   
                 2 = C or optional 
               
               
                   
                   
                   
                 t_BB_dC or 
               
               
                   
                   
                   
                 N4_Et_dC 
               
               
                   
               
               
                 338 
                 ESR1_V533M_RS02 
                 GGTCATAGAG1GGCACCCT 
                 1 = Optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 339 
                 ESR1_V533M_RS20 
                 GGTCATAGAG1GGCACC2T 
                 1 = Optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
                   
                   
                 2 = C or optional 
               
               
                   
                   
                   
                 LNA-A 
               
               
                   
               
               
                 340 
                 ESR1_V533M_RS03 
                 GGTCATAGAG1GGCACCGT 
                 1 = Optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 341 
                 ESR1_V533M_RS04 
                 GGTCATAGAG1GGCACCTT 
                 1 = Optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 342 
                 ESR1_V533M_RS05 
                 GGTCATAGAG1GGCACAAT 
                 1 = Optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 343 
                 ESR1_V533M_RS06 
                 GGTCATAGAG1GGCACGAT 
                 1 = Optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 344 
                 ESR1_V533M_RS07 
                 GGTCATAGAG1GGCACTAT 
                 1 = Optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 345 
                 ESR1_V533M_RS08 
                 GGTCATAGAG1GGCAACAT 
                 1 = Optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 346 
                 ESR1_V533M_RS09 
                 GGTCATAGAG1GGCAGCAT 
                 1 = Optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 347 
                 ESR1_V533M_RS10 
                 GGTCATAGAG1GGCATCAT 
                 1 = Optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 348 
                 ESR1_V533M_RS11 
                 AGGTCATAGAG1GGCAGCAT 
                 1 = Optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 349 
                 ESR1_V533M_RS12 
                 AGGTCATAGAG1GGCTCCAT 
                 1 = Optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 350 
                 ESR1_V533M_RS13 
                 AGGTCATAGAG1GGCGCCAT 
                 1 = Optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 351 
                 ESR1_V533M_RS14 
                 AGGTCATAGAG1GGCCCCAT 
                 1 = Optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 352 
                 ESR1_V533M_RS15 
                 CAGGTCATAGAG1GGCTCCAT 
                 1 = Optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 353 
                 ESR1_V533M_RS16 
                 CAGGTCATAGAG1GGCGCCAT 
                 1 = Optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 354 
                 ESR1_V533M_RS17 
                 CAGGTCATAGAG1GGCCCCAT 
                 1 = Optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 355 
                 ESR1_V533M_TBBA_FS01 
                 ACAGCATGAAGTGCAAG1ACA 
                 1 = A or optional 
               
               
                   
                   
                   
                 t_BB_dA 
               
               
                   
               
               
                 356 
                 ESR1_V533M_TBBDC_FS01 
                 ACAGCATGAAGTGCAAGAA1A 
                 1 = C or optional 
               
               
                   
                   
                   
                 t_BB_dC 
               
               
                   
               
               
                 357 
                 ESR1_V534E_CF01 
                 CTTTCTGTGTCTTCCCACCTAC 
                   
               
               
                   
               
               
                 358 
                 ESR1_V534E_CRO1 
                 GCTTTGGTCCGTCTCCT 
                   
               
               
                   
               
               
                 359 
                 ESR1_V534E_CR02 
                 TGGCTTTGGTCCGTCTCCT 
                   
               
               
                   
               
               
                 360 
                 ESR1_V534E_CR03 
                 ATGTAGGCGGTGGGCGTC 
                   
               
               
                   
               
               
                 361 
                 ESR1_V534E_CR04 
                 CTCCACGGCTAGTGGGCG 
                   
               
               
                   
               
               
                 362 
                 ESR1_V534E_CR05 
                 TGCCCCTCCACGGCTAGT 
                   
               
               
                   
               
               
                 363 
                 ESR1_V534E_F_P01 
                 1TCTATGA2CCTGCTGCTGGAGATGCTGGA3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phophate 
               
               
                   
               
               
                 364 
                 ESR1_V534E_FS01 
                 GCATGAAGTGCAAGAACGTGG1 
                 1 = A or optional 
               
               
                   
                   
                   
                 N6_Bz_dA 
               
               
                   
               
               
                 365 
                 ESR1_V534E_FS02 
                 GCATGAAGTGCAAGAACGTGAA 
                   
               
               
                   
               
               
                 366 
                 ESR1_V534E_FS03 
                 GCATGAAGTGCAAGAACGTGCA 
                   
               
               
                   
               
               
                 367 
                 ESR1_V534E_FS04 
                 GCATGAAGTGCAAGAACGTGTA 
                   
               
               
                   
               
               
                 368 
                 ESR1_V534E_FS05 
                 GCATGAAGTGCAAGAACGTAGA 
                   
               
               
                   
               
               
                 369 
                 ESR1_V534E_FS06 
                 GCATGAAGTGCAAGAACGTCGA 
                   
               
               
                   
               
               
                 370 
                 ESR1_V534E_FS07 
                 GCATGAAGTGCAAGAACGTTGA 
                   
               
               
                   
               
               
                 371 
                 ESR1_V534E_FS08 
                 GCATGAAGTGCAAGAACGAGGA 
                   
               
               
                   
               
               
                 372 
                 ESR1_V534E_FS09 
                 GCATGAAGTGCAAGAACGCGGA 
                   
               
               
                   
               
               
                 373 
                 ESR1_V534E_FS10 
                 GCATGAAGTGCAAGAACGGGGA 
                   
               
               
                   
               
               
                 374 
                 ESR1_V534E_R_P01 
                 1TGCACTT2CATGCTGTACAGATGCTCCATG3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 375 
                 ESR1_V534E_R_P03 
                 1TGCACTT2CA3GCTG3ACAGATGC3CCATG4 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = pdU(3x) 
               
               
                   
                   
                   
                 4 = Phosphate 
               
               
                   
               
               
                 376 
                 ESR1_V534E_R_P04 
                 1TGCACTT2CATGCTGTACAGATGCTCCAT3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 377 
                 ESR1_V534E_R_P05 
                 1TGCACTT2CA3GCTG3ACAGA3GCTCCA34 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = pdU(4x) 
               
               
                   
                   
                   
                 4 = Phosphate 
               
               
                   
               
               
                 378 
                 ESR1_V534E_RS01 
                 GCAGGTCATAGAG1GGCT 
                 1 = G or optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 379 
                 ESR1_V534E_RS02 
                 GCAGGTCATAGAGGGGAT 
                   
               
               
                   
               
               
                 380 
                 ESR1_V534E_RS03 
                 GCAGGTCATAGAGGGGGT 
                   
               
               
                   
               
               
                 381 
                 ESR1_V534E_RS04 
                 GCAGGTCATAGAGGGGTT 
                   
               
               
                   
               
               
                 382 
                 ESR1_V534E_RS05 
                 GCAGGTCATAGAGGGACT 
                   
               
               
                   
               
               
                 383 
                 ESR1_V534E_RS06 
                 GCAGGTCATAGAGGGCCT 
                   
               
               
                   
               
               
                 384 
                 ESR1_V534E_RS07 
                 GCAGGTCATAGAGGGTCT 
                   
               
               
                   
               
               
                 385 
                 ESR1_V534E_RS08 
                 GCAGGTCATAGAGGAGCT 
                   
               
               
                   
               
               
                 386 
                 ESR1_V534E_RS09 
                 GCAGGTCATAGAGGCGCT 
                   
               
               
                   
               
               
                 387 
                 ESR1_V534E_RS10 
                 GCAGGTCATAGAGGTGCT 
                   
               
               
                   
               
               
                 388 
                 ESR1_V534E_RS11 
                 GCAGGTCATAGAG1GGCT 
                 1 = G or optional 
               
               
                   
                   
                   
                 7_Dz_dG 
               
               
                   
               
               
                 389 
                 ESR1_V534E_RS13 
                 GCAGGTCATAGAGTGGCT 
                   
               
               
                   
               
               
                 390 
                 ESR1_V534E_RS14 
                 GCAGGTCATAGAGCGGCT 
                   
               
               
                   
               
               
                 391 
                 ESR1_V534E_RS15 
                 CAGCAGGTCATAGAGAGGCT 
                   
               
               
                   
               
               
                 392 
                 ESR1_V534E_RS16 
                 CAGCAGGTCATAGAGTGGCT 
                   
               
               
                   
               
               
                 393 
                 ESR1_V534E_RS17 
                 CAGCAGGTCATAGAGCGGCT 
                   
               
               
                   
               
               
                 394 
                 ESR1_Y537C_CF01 
                 CTGTGTCTTCCCACCTACAGTA 
                   
               
               
                   
               
               
                 395 
                 ESR1_Y537C_CR01 
                 AAGTGGCTTTGGTCCGT 
                   
               
               
                   
               
               
                 396 
                 ESR1_Y537C_F_P01 
                 1TACATG2CGCCCACTAGCCGTGGA3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 397 
                 ESR1_Y537C_FS01 
                 ACGTGGTGCCCCTCTG 
                   
               
               
                   
               
               
                 398 
                 ESR1_Y537C_FS02 
                 ACGTGGTGCCCCTCAG 
                   
               
               
                   
               
               
                 399 
                 ESR1_Y537C_FS03 
                 ACGTGGTGCCCCTCCG 
                   
               
               
                   
               
               
                 400 
                 ESR1_Y537C_FS04 
                 ACGTGGTGCCCCTCGG 
                   
               
               
                   
               
               
                 401 
                 ESR1_Y537C_FS05 
                 ACGTGGTGCCCCTATG 
                   
               
               
                   
               
               
                 402 
                 ESR1_Y537C_FS06 
                 ACGTGGTGCCCCTGTG 
                   
               
               
                   
               
               
                 403 
                 ESR1_Y537C_FS07 
                 ACGTGGTGCCCCTTTG 
                   
               
               
                   
               
               
                 404 
                 ESR1_Y537C_FS08 
                 ACGTGGTGCCCCACTG 
                   
               
               
                   
               
               
                 405 
                 ESR1_Y537C_FS09 
                 ACGTGGTGCCCCCCTG 
                   
               
               
                   
               
               
                 406 
                 ESR1_Y537C_FS10 
                 ACGTGGTGCCCCGCTG 
                   
               
               
                   
               
               
                 407 
                 ESR1_Y537C_R_P01 
                 1TGCACTT2CATGCTGTACAGATGCTCCATGC3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 408 
                 ESR1_Y537C_RS01 
                 CTCCAGCAGCAGGT1AC 
                 1 = C or optional 
               
               
                   
                   
                   
                 t_BB_dC 
               
               
                   
               
               
                 409 
                 ESR1_Y537C_RS02 
                 CTCCAGCAGCAGGTCCC 
                   
               
               
                   
               
               
                 410 
                 ESR1_Y537C_RS03 
                 CTCCAGCAGCAGGTCGC 
                   
               
               
                   
               
               
                 411 
                 ESR1_Y537C_RS04 
                 CTCCAGCAGCAGGTCTC 
                   
               
               
                   
               
               
                 412 
                 ESR1_Y537C_RS05 
                 CTCCAGCAGCAGGTAAC 
                   
               
               
                   
               
               
                 413 
                 ESR1_Y537C_RS06 
                 CTCCAGCAGCAGGTGAC 
                   
               
               
                   
               
               
                 414 
                 ESR1_Y537C_RS07 
                 CTCCAGCAGCAGGTTAC 
                   
               
               
                   
               
               
                 415 
                 ESR1_Y537C_RS08 
                 CTCCAGCAGCAGGACAC 
                   
               
               
                   
               
               
                 416 
                 ESR1_Y537C_RS09 
                 CTCCAGCAGCAGGCCAC 
                   
               
               
                   
               
               
                 417 
                 ESR1_Y537C_RS10 
                 CTCCAGCAGCAGGGCAC 
                   
               
               
                   
               
               
                 418 
                 ESR1_Y537C_RS11 
                 ATCTCCAGCAGCAGGACAC 
                   
               
               
                   
               
               
                 419 
                 ESR1_Y537C_RS12 
                 CATCTCCAGCAGCAGGACAC 
                   
               
               
                   
               
               
                 420 
                 ESR1_Y537C_RS13 
                 ATCTCCAGCAGCAGGT1AC 
                 1 = C or optional 
               
               
                   
                   
                   
                 t_BB_dC 
               
               
                   
               
               
                 421 
                 ESR1_Y537C_RS14 
                 ATCTCCAGCAGCAGGTC1C 
                 1 = A or optional 
               
               
                   
                   
                   
                 t_BB_dA 
               
               
                   
               
               
                 422 
                 ESR1_Y537C_RS15 
                 ATCTCCAGCAGCAGGTCA1 
                 1 = C or optional 
               
               
                   
                   
                   
                 t_BB_dC 
               
               
                   
               
               
                 423 
                 ESR1_Y537C_RS16 
                 CTCCAGCAGCAGGT1AC 
                 1 = C or optional 
               
               
                   
                   
                   
                 t_BB_dC 
               
               
                   
               
               
                 424 
                 ESR1_Y537C_RS17 
                 CTCCAGCAGCAGGTC1C 
                 1 = A or optional 
               
               
                   
                   
                   
                 t_BB_dA or 
               
               
                   
                   
                   
                 LNA-A 
               
               
                   
               
               
                 425 
                 ESR1_Y537C_RS18 
                 CTCCAGCAGCAGGTCA1 
                 1 = C or optional 
               
               
                   
                   
                   
                 t_BB_dC 
               
               
                   
               
               
                 426 
                 ESR1_Y537N_FS01 
                 AACGTGGTGCCCCTCA 
                   
               
               
                   
               
               
                 427 
                 ESR1_Y537N_FS02 
                 AACGTGGTGCCCCTAA 
                   
               
               
                   
               
               
                 428 
                 ESR1_Y537N_FS03 
                 AACGTGGTGCCCCTGA 
                   
               
               
                   
               
               
                 429 
                 ESR1_Y537N_FS04 
                 AACGTGGTGCCCCTTA 
                   
               
               
                   
               
               
                 430 
                 ESR1_Y537N_FS05 
                 AACGTGGTGCCCCACA 
                   
               
               
                   
               
               
                 431 
                 ESR1_Y537N_FS06 
                 AACGTGGTGCCCCCCA 
                   
               
               
                   
               
               
                 432 
                 ESR1_Y537N_FS07 
                 AACGTGGTGCCCCGCA 
                   
               
               
                   
               
               
                 433 
                 ESR1_Y537N_FS08 
                 AACGTGGTGCCCATCA 
                   
               
               
                   
               
               
                 434 
                 ESR1_Y537N_FS09 
                 AACGTGGTGCCCGTCA 
                   
               
               
                   
               
               
                 435 
                 ESR1_Y537N_FS10 
                 AACGTGGTGCCCTTCA 
                   
               
               
                   
               
               
                 436 
                 ESR1_Y537N_RS01 
                 CTCCAGCAGCAGGT1ATT 
                 1 = C or optional 
               
               
                   
                   
                   
                 t_BB_dC 
               
               
                   
               
               
                 437 
                 ESR1_Y537N_RS02 
                 CTCCAGCAGCAGGTCAAT 
                   
               
               
                   
               
               
                 438 
                 ESR1_Y537N_RS03 
                 CTCCAGCAGCAGGTCACT 
                   
               
               
                   
               
               
                 439 
                 ESR1_Y537N_RS04 
                 CTCCAGCAGCAGGTCAGT 
                   
               
               
                   
               
               
                 440 
                 ESR1_Y537N_RS05 
                 CTCCAGCAGCAGGTCCTT 
                   
               
               
                   
               
               
                 441 
                 ESR1_Y537N_RS06 
                 CTCCAGCAGCAGGTCGTT 
                   
               
               
                   
               
               
                 442 
                 ESR1_Y537N_RS07 
                 CTCCAGCAGCAGGTCTTT 
                   
               
               
                   
               
               
                 443 
                 ESR1_Y537N_RS08 
                 CTCCAGCAGCAGGTAATT 
                   
               
               
                   
               
               
                 444 
                 ESR1_Y537N_RS09 
                 CTCCAGCAGCAGGTGATT 
                   
               
               
                   
               
               
                 445 
                 ESR1_Y537N_RS10 
                 CTCCAGCAGCAGGTTATT 
                   
               
               
                   
               
               
                 446 
                 ESR1_Y537N_RS11 
                 ATCTCCAGCAGCAGGTTATT 
                   
               
               
                   
               
               
                 447 
                 ESR1_Y537N_RS12 
                 CATCTCCAGCAGCAGGTTATT 
                   
               
               
                   
               
               
                 448 
                 ESR1_Y537N_RS13 
                 GCATCTCCAGCAGCAGGTTATT 
                   
               
               
                   
               
               
                 449 
                 ESR1_Y537S_FS01 
                 ACGTGGTGCCCCTCTC 
                   
               
               
                   
               
               
                 450 
                 ESR1_Y537S_FS02 
                 ACGTGGTGCCCCTCAC 
                   
               
               
                   
               
               
                 451 
                 ESR1_Y537S_FS03 
                 ACGTGGTGCCCCTCCC 
                   
               
               
                   
               
               
                 452 
                 ESR1_Y537S_FS04 
                 ACGTGGTGCCCCTCGC 
                   
               
               
                   
               
               
                 453 
                 ESR1_Y537S_FS05 
                 ACGTGGTGCCCCTATC 
                   
               
               
                   
               
               
                 454 
                 ESR1_Y537S_FS06 
                 ACGTGGTGCCCCTGTC 
                   
               
               
                   
               
               
                 455 
                 ESR1_Y537S_FS07 
                 ACGTGGTGCCCCTTTC 
                   
               
               
                   
               
               
                 456 
                 ESR1_Y537S_FS08 
                 ACGTGGTGCCCCACTC 
                   
               
               
                   
               
               
                 457 
                 ESR1_Y537S_FS09 
                 ACGTGGTGCCCCCCTC 
                   
               
               
                   
               
               
                 458 
                 ESR1_Y537S_FS10 
                 ACGTGGTGCCCCGCTC 
                   
               
               
                   
               
               
                 459 
                 ESR1_Y537S_RS01 
                 ATCTCCAGCAGCAGGT1AG 
                 1 = C or optional 
               
               
                   
                   
                   
                 t_BB_dC 
               
               
                   
               
               
                 460 
                 ESR1_Y537S_RS02 
                 ATCTCCAGCAGCAGGTCCG 
                   
               
               
                   
               
               
                 461 
                 ESR1_Y537S_RS03 
                 ATCTCCAGCAGCAGGTCGG 
                   
               
               
                   
               
               
                 462 
                 ESR1_Y537S_RS04 
                 ATCTCCAGCAGCAGGTCTG 
                   
               
               
                   
               
               
                 463 
                 ESR1_Y537S_RS05 
                 ATCTCCAGCAGCAGGTAAG 
                   
               
               
                   
               
               
                 464 
                 ESR1_Y537S_RS06 
                 ATCTCCAGCAGCAGGTGAG 
                   
               
               
                   
               
               
                 465 
                 ESR1_Y537S_RS07 
                 ATCTCCAGCAGCAGGTTAG 
                   
               
               
                   
               
               
                 466 
                 ESR1_Y537S_RS08 
                 ATCTCCAGCAGCAGGACAG 
                   
               
               
                   
               
               
                 467 
                 ESR1_Y537S_RS09 
                 ATCTCCAGCAGCAGGCCAG 
                   
               
               
                   
               
               
                 468 
                 ESR1_Y537S_RS10 
                 ATCTCCAGCAGCAGGGCAG 
                   
               
               
                   
               
               
                 469 
                 ESR1_Y537S_RS11 
                 CATCTCCAGCAGCAGGACAG 
                   
               
               
                   
               
               
                 470 
                 ESR1_Y537S_RS12 
                 CATCTCCAGCAGCAGGGCAG 
                   
               
               
                   
               
               
                 471 
                 ESR1_Y537S_RS13 
                 GCATCTCCAGCAGCAGGACAG 
                   
               
               
                   
               
               
                 472 
                 ESR1_Y537S_RS14 
                 CTCCAGCAGCAGGGCAG 
                   
               
               
                   
               
               
                 473 
                 ESR1K303RWSNP_CF01 
                 AGAGATGATGGGGAGGGCA 
                   
               
               
                   
               
               
                 474 
                 ESR1K303RWSNP_CF02 
                 AGATGATGGGGAGGGCA 
                   
               
               
                   
               
               
                 475 
                 ESR1K303RWSNP_CR01 
                 TCAGCATCCAACAAGGCA 
                   
               
               
                   
               
               
                 476 
                 ESR1K303RWSNP_CR02 
                 CTCAGCATCCAACAAGGCA 
                   
               
               
                   
               
               
                 477 
                 ESR1K303RWSNP_F_P01 
                 1TTGTCCCTGAC2GGCCGACCAGATGGTCA3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 478 
                 ESR1K303RWSNP_FS01 
                 CGCTCATGATCAAACGCTCTAAGAG 
                   
               
               
                   
               
               
                 479 
                 ESR1K303RWSNP_FS02 
                 CGCTCATGATCAAACGCTCTAAGCG 
                   
               
               
                   
               
               
                 480 
                 ESR1K303RWSNP_FS03 
                 CGCTCATGATCAAACGCTCTAAGGG 
                   
               
               
                   
               
               
                 481 
                 ESR1K303RWSNP_FS04 
                 CGCTCATGATCAAACGCTCTAAGTG 
                   
               
               
                   
               
               
                 482 
                 ESR1K303RWSNP_FS05 
                 CGCTCATGATCAAACGCTCTAAAAG 
                   
               
               
                   
               
               
                 483 
                 ESR1K303RWSNP_FS06 
                 CGCTCATGATCAAACGCTCTAACAG 
                   
               
               
                   
               
               
                 484 
                 ESR1K303RWSNP_FS07 
                 CGCTCATGATCAAACGCTCTAATAG 
                   
               
               
                   
               
               
                 485 
                 ESR1K303RWSNP_FS08 
                 CGCTCATGATCAAACGCTCTACGAG 
                   
               
               
                   
               
               
                 486 
                 ESR1K303RWSNP_FS09 
                 CGCTCATGATCAAACGCTCTAGGAG 
                   
               
               
                   
               
               
                 487 
                 ESR1K303RWSNP_FS10 
                 CGCTCATGATCAAACGCTCTATGAG 
                   
               
               
                   
               
               
                 488 
                 ESR1K303RWSNP_R_P01 
                 1TTTGATCATGA2GCGGGCTTGGCCAAAGGTT3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 489 
                 ESR1K303RWSNP_RS01 
                 ACAAGGCCAGGCTGTTCC 
                   
               
               
                   
               
               
                 490 
                 ESR1K303RWSNP_RS02 
                 ACAAGGCCAGGCTGTTAC 
                   
               
               
                   
               
               
                 491 
                 ESR1K303RWSNP_RS03 
                 ACAAGGCCAGGCTGTTGC 
                   
               
               
                   
               
               
                 492 
                 ESR1K303RWSNP_RS04 
                 ACAAGGCCAGGCTGTTTC 
                   
               
               
                   
               
               
                 493 
                 ESR1K303RWSNP_RS05 
                 ACAAGGCCAGGCTGTACC 
                   
               
               
                   
               
               
                 494 
                 ESR1K303RWSNP_RS06 
                 ACAAGGCCAGGCTGTCCC 
                   
               
               
                   
               
               
                 495 
                 ESR1K303RWSNP_RS07 
                 ACAAGGCCAGGCTGTGCC 
                   
               
               
                   
               
               
                 496 
                 ESR1K303RWSNP_RS08 
                 ACAAGGCCAGGCTGATCC 
                   
               
               
                   
               
               
                 497 
                 ESR1K303RWSNP_RS09 
                 ACAAGGCCAGGCTGCTCC 
                   
               
               
                   
               
               
                 498 
                 ESR1K303RWSNP_RS10 
                 ACAAGGCCAGGCTGGTCC 
                   
               
               
                   
               
               
                 499 
                 SC_ESR1_D01F 
                 GTCTGGCGAGAGATGCAAA 
                   
               
               
                   
               
               
                 500 
                 SC_ESR1_D01FP1 
                 1CTCTAC2TTTCCTTACCTCCTTCCTTCCA3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 501 
                 SC_ESR1_D01R 
                 GCCTCAATGAAGACAACTTGAA 
                   
               
               
                   
               
               
                 502 
                 SC_ESR1_D02F 
                 AGGATAAAGTGGATCTGCTGCA 
                   
               
               
                   
               
               
                 503 
                 SC_ESR1_D02R 
                 CCTGGCGTCGATTATCTGAA 
                   
               
               
                   
               
               
                 504 
                 SC_ESR1_D03F 
                 GCTGTTAATTGTCCATGCATAA 
                   
               
               
                   
               
               
                 505 
                 SC_ESR1_D03R 
                 GAAAGGGGAGAACAAGCTAAA 
                   
               
               
                   
               
               
                 506 
                 SC_ESR1_D04F 
                 GAGGAATGGATTTCAATGGAA 
                   
               
               
                   
               
               
                 507 
                 SC_ESR1_D04R 
                 CCCTGGGTCTGTGATCACTAA 
                   
               
               
                   
               
               
                 508 
                 SC_ESR1EX1WTASR1 
                 GCCACGGACCATGACCATGA 
                   
               
               
                   
               
               
                 509 
                 SC_ESR1EX1WTCRP1 
                 CTTGAGCTGCGGACGGTTCA 
                   
               
               
                   
               
               
                 510 
                 SC_ESR1EX1WTPRB1 
                 1TGGCCCTA2CTGCATCAGATCCAAGG3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 511 
                 SC_ESR1EX2WTASR1 
                 CAGAGAAAGATTGGCCAGTACC 
                   
               
               
                   
               
               
                 512 
                 SC_ESR1EX2WTASR2 
                 GCCAGTACCAATGACAAGGGAAG 
                   
               
               
                   
               
               
                 513 
                 SC_ESR1EX2WTASR3 
                 CCAGGGTGGCAGAGAAAGATT 
                   
               
               
                   
               
               
                 514 
                 SC_ESR1EX2WTCRP1 
                 CAGACTCCATAATGGTAGCCTGA 
                   
               
               
                   
               
               
                 515 
                 SC_ESR1EX2WTCRP2 
                 TAATGGTAGCCTGAAGCATAGTCAT 
                   
               
               
                   
               
               
                 516 
                 SC_ESR1EX2WTCRP3 
                 CACTGCACAGTAGCGAGTCTCCT 
                   
               
               
                   
               
               
                 517 
                 SC_ESR1EX2WTPRB1 
                 1TGACAAGG2GAAGTATGGCTATGGAATCT3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 518 
                 SC_ESR1EX2WTPRB2 
                 1TGACAAG2G3AAGTATGGCTATGGAATCT4 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = 7_Dz_dG 
               
               
                   
                   
                   
                 3 = Quencher 
               
               
                   
                   
                   
                 4 = Phosphate 
               
               
                   
               
               
                 519 
                 SC_ESR1EX2WTPRB2B 
                 1CTATGGAA2TCTGCCAAGGAGACTCGCTA3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 520 
                 SC_ESR1EX2WTPRB3 
                 1TGACAAGGGA2AGTATGGCTATGGAATCT3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 521 
                 SC_ESR1EX2WTPRB3B 
                 1CAGTACCA2ATGACAAGGGAAGTATGGCT3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 522 
                 SC_ESR1EX2WTPRB4 
                 1TGACAAGG2GAAGTATGGCTATGGAATCT3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 523 
                 SC_ESR1EX5E380QASR1 
                 CATGATCAGGTCCACCTTCTAC 
                   
               
               
                   
               
               
                 524 
                 SC_ESR1EX5E380QASR2 
                 CCATGATCAGGTCCACCTTCTAC 
                   
               
               
                   
               
               
                 525 
                 SC_ESR1EX5E380QASR3 
                 TCCATGATCAGGTCCACCTTCTAC 
                   
               
               
                   
               
               
                 526 
                 SC_ESR1EX5E380QCRP1 
                 GAGCAAGTTAGGAGCAAACAGTA 
                   
               
               
                   
               
               
                 527 
                 SC_ESR1EX5E380QCRP2 
                 AGAGCAAGTTAGGAGCAAACAGTA 
                   
               
               
                   
               
               
                 528 
                 SC_ESR1EX5E380QCRP3 
                 AAGAGCAAGTTAGGAGCAAACAGTA 
                   
               
               
                   
               
               
                 529 
                 SC_ESR1EX5WTASR1 
                 CATGATCAGGTCCACCTTCTAG 
                   
               
               
                   
               
               
                 530 
                 SC_ESR1EX5WTCRP1 
                 GAGCAAGTTAGGAGCAAACAGTA 
                   
               
               
                   
               
               
                 531 
                 SC_ESR1EX5WTCRP2 
                 AGAGCAAGTTAGGAGCAAACAGTA 
                   
               
               
                   
               
               
                 532 
                 SC_ESR1EX5WTCRP3 
                 AAGAGCAAGTTAGGAGCAAACAGTA 
                   
               
               
                   
               
               
                 533 
                 SC_ESR1EX5WTPBR1 
                 1TGCCTG2GCTAGAGATCCTGATGATTGGT3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 534 
                 SC_ESR1EX6L429DVASR1 
                 GGTAGAGATCTTCGACATGCTGG 
                   
               
               
                   
               
               
                 535 
                 SC_ESR1EX6L429DVASR2 
                 ATGGTAGAGATCTTCGACATGCTGG 
                   
               
               
                   
               
               
                 536 
                 SC_ESR1EX6V422DVASR1 
                 ATGTGTAGAGGGCATGGAGATCT 
                   
               
               
                   
               
               
                 537 
                 SC_ESR1EX6V422DVCRP1 
                 GTTATCAACTCACCAGAATTAAGCAA 
                   
               
               
                   
               
               
                 538 
                 SC_ESR1EX6V422DVCRP2 
                 TGTTATCAACTCACCAGAATTAAGCAA 
                   
               
               
                   
               
               
                 539 
                 SC_ESR1EX6V422DVCRP3 
                 GTGTTATCAACTCACCAGAATTAAGCAA 
                   
               
               
                   
               
               
                 540 
                 SC_ESR1EX6V422DVPRB1 
                 1CATCTCGGTT2CCGCATGATGAATCTGC3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 541 
                 SC_ESR1EX6WT2ASR1 
                 GGTAGAGATCTTCGACATGCTGC 
                   
               
               
                   
               
               
                 542 
                 SC_ESR1EX6WT2CRP1 
                 GTTATCAACTCACCAGAATTAAGCAA 
                   
               
               
                   
               
               
                 543 
                 SC_ESR1EX6WTASR1 
                 GGTAGAGATCTTCGACATGCTGC 
                   
               
               
                   
               
               
                 544 
                 SC_ESR1EX6WTCRP1 
                 TCACCAGAATTAAGCAAAATAATAGATT 
                   
               
               
                   
               
               
                 545 
                 SC_ESR1EX6WTPRB1 
                 1CATCTCGGTT2CCGCATGATGAATCTGCA3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 546 
                 SC_ESR1EX7S463PASR1 
                 ATTCAGGAGTGTACACATTTCTGC 
                   
               
               
                   
               
               
                 547 
                 SC_ESR1EX7WTASR1 
                 CATTCAGGAGTGTACACATTTCTGT 
                   
               
               
                   
               
               
                 548 
                 SC_ESR1EX7WTCRP1 
                 ATCAGGTGGATCAAAGTGTCTGT 
                   
               
               
                   
               
               
                 549 
                 SC_ESR1EX7WTPRB1 
                 1TCTCTG2GAAGAGAAGGACCATATCCACC3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 550 
                 SC_ESR1EX8D538GASR1 
                 ACGTGGTGCCCCTCTATGG 
                   
               
               
                   
               
               
                 551 
                 SC_ESR1EX8L536HASR1 
                 CAAGAACGTGGTGCCCCA 
                   
               
               
                   
               
               
                 552 
                 SC_ESR1EX8L536HASR2 
                 TGCAAGAACGTGGTGCCCCA 
                   
               
               
                   
               
               
                 553 
                 SC_ESR1EX8L536HASR3 
                 GTGCAAGAACGTGGTGCCCCA 
                   
               
               
                   
               
               
                 554 
                 SC_ESR1EX8L536HASR4 
                 GTGCAAGAACGTGGTGCCACA 
                   
               
               
                   
               
               
                 555 
                 SC_ESR1EX8L536PASR1 
                 CAAGAACGTGGTGCCCCC 
                   
               
               
                   
               
               
                 556 
                 SC_ESR1EX8L536QASR1 
                 CAAGAACGTGGTGCCCCAG 
                   
               
               
                   
               
               
                 557 
                 SC_ESR1EX8L536RASR1 
                 CAAGAACGTGGTGCCCCG 
                   
               
               
                   
               
               
                 558 
                 SC_ESR1EX8P535H1ASR1 
                 TGAAGTGCAAGAACGTGGTGCA 
                   
               
               
                   
               
               
                 559 
                 SC_ESR1EX8P535H1ASR2 
                 CATGAAGTGCAAGAACGTGGTGCA 
                   
               
               
                   
               
               
                 560 
                 SC_ESR1EX8P535H2ASR1 
                 TGAAGTACAAGAACGTGGTGCA 
                   
               
               
                   
               
               
                 561 
                 SC_ESR1EX8P535H2ASR2 
                 ATGAAGTACAAGAACGTGGTGCA 
                   
               
               
                   
               
               
                 562 
                 SC_ESR1EX8P535H2ASR3 
                 CATGAAGTACAAGAACGTGGTGCA 
                   
               
               
                   
               
               
                 563 
                 SC_ESR1EX8P535H2ASR4 
                 TGAAGTGCAAGAACGTGATGCA 
                   
               
               
                   
               
               
                 564 
                 SC_ESR1EX8P535H2ASR5 
                 AGCATGAAGTACAAGAACGTGGTGCA 
                   
               
               
                   
               
               
                 565 
                 SC_ESR1EX8P535H3ASR1 
                 TGAAGTGCAAGAACGTGGTACA 
                   
               
               
                   
               
               
                 566 
                 SC_ESR1EX8P535H3ASR2 
                 ATGAAGTGCAAGAACGTGGTACA 
                   
               
               
                   
               
               
                 567 
                 SC_ESR1EX8P535H3ASR3 
                 CATGAAGTGCAAGAACGTGGTACA 
                   
               
               
                   
               
               
                 568 
                 SC_ESR1EX8V534EASR1 
                 CATGAAGTGCAAGAACGTGGA 
                   
               
               
                   
               
               
                 569 
                 SC_ESR1EX8V534EASR2 
                 GCATGAAGTGCAAGAACGTGGA 
                   
               
               
                   
               
               
                 570 
                 SC_ESR1EX8V534EASR3 
                 AGCATGAAGTGCAAGAACGTGGA 
                   
               
               
                   
               
               
                 571 
                 SC_ESR1EX8WTASR1 
                 ACGTGGTGCCCCTCTATGAC 
                   
               
               
                   
               
               
                 572 
                 SC_ESR1EX8WTCRP1 
                 GCTTTGGTCCGTCTCCTCC 
                   
               
               
                   
               
               
                 573 
                 SC_ESR1EX8WTPRB1 
                 1CTGCTG2GAGATGCTGGACGCCCACC3 
                 1 = Reporter 
               
               
                   
                   
                   
                 2 = Quencher 
               
               
                   
                   
                   
                 3 = Phosphate 
               
               
                   
               
               
                 574 
                 SC_ESR1EX8Y537CASR1 
                 AGAACGTGGTGCCCCTCTG 
                   
               
               
                   
               
               
                 575 
                 SC_ESR1EX8Y537NASR1 
                 AGAACGTGGTGCCCCTCA 
                   
               
               
                   
               
               
                 576 
                 SC_ESR1EX8Y537NASR2 
                 AAGAACGTGGTGCCCCTCA 
                   
               
               
                   
               
               
                 577 
                 SC_ESR1EX8Y537NASR3 
                 CAAGAACGTGGTGCCCCTCA 
                   
               
               
                   
               
               
                 578 
                 SC_ESR1EX8Y537SASR1 
                 AGAACGTGGTGCCCCTCTC 
                   
               
               
                   
               
               
                 579 
                 ESR1_S463P_CF02 
                 CTCCTAGACCTCATCCTCTTTGA 
               
               
                   
               
            
           
         
       
     
     Oligonucleotide Selection for High Sensitivity and Specificity Detection of Mutants 
     Primers were selected to ensure that mutant signal could be sensitively and specifically detected compared to wild type. A primer pair and probe designed to be specific for the ESR1 K303R mutation and for the L536_D538&gt;P mutation were used detect mutant template added to wild type DNA at a ratio of 1:1600 (100 copies of mutant DNA in 160,000 copies of wild type) and in wild type DNA alone. Allele specific amplification and detection were carried out on the cobas z 480. The data are expressed in Ct curves plotting amplification cycles vs fluorescence signal (indicating detection of the amplification product) 
       FIG. 1A  shows a reaction specific for K303R with no discrimination. The selected primers and probe amplify and detect signal from the mutant and wild type samples at essentially the same level.  FIG. 1B  shows a reaction specific for K303R with better discrimination. The Ct&#39;s of the two wild type samples are shifted to the right by at least 2 cycles.  FIG. 1C  shows a much more specific reaction for L536_D538&gt;P that is also sensitive enough to detect mutant DNA at a ratio of at least 1:1600. The wild type samples are not detected at all, while the mutant samples have a Ct around 35-38. 
     A similar comparison is shown in  FIG. 2A ,  FIG. 2B , and  FIG. 2C  with primers and probes designed to be specific for the L536R mutation.  FIG. 2A  shows a reaction with no discrimination using the L536R_RS01 allele-specific primer. Wild type is amplified at essentially the same level as mutant.  FIG. 2B  shows a reaction with better discrimination using the L536R_RS09 allele-specific primer, where wild type has a Ct delayed by at least 5 cycles compared to mutant.  FIG. 2C  shows a reaction with good discrimination using the L536R_RS04 allele-specific primer. Wild type has a Ct over 50 cycles, considered not detected, compared to a mutant Ct well in the detectable range around 35. 
     Specificity of Multiplex Mutation Detection 
     The primer pair and probe sets were tested in multiplex to determine specificity and ensure that they would not detect other ESR1 mutations. Separate sample mixtures were prepared for each of the 18 mutants listed in Table 1, with mutant DNA added 1:1 with wild type (5000 copies of each). Primer pairs and probes specific for all 18 mutations were added.  FIG. 3  shows an exemplary Ct curve with high specificity for ESR1 P535H. The two left-most curves with a Ct of around 25-30 represent specific detection of the ESR1 P535H mutation present in the sample (indicated with light arrow). The Ct&#39;s for the other primer pair and probe sets have a much more delayed Ct (indicated between two black arrows). 
     Assay Linearity 
     Linearity of detection was also detected across a range of template concentrations for each mutation. As an example primer pair and probe specific for ESR1 S341L were used to amplify 5-5000 copies mutant template in a background of 10,000 wild type copies. The results are shown in Table 3, and show that the assay is highly linear. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 3 
               
               
                   
                   
               
               
                   
                 Mutant copy number 
                 Ct 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 5 
                 33.04 
               
               
                   
                 50 
                 29.32 
               
               
                   
                 500 
                 26.23 
               
               
                   
                 5000 
                 22.89 
               
               
                   
                 Slope 
                 −3.36 
               
               
                   
                 R 2   
                 0.9987 
               
               
                   
                   
               
            
           
         
       
     
     Detection of ESR1 Mutations in Contrived Plasma Samples 
     The assay was tested in plasma background to determine if plasma components would interfere. Mutant DNA was added at 1000 copies into 2 ml normal (wild type) plasma. DNA was extracted with the cobas° DNA Sample Preparation Kit. DNA equivalent to that from 0.5 ml plasma was added to each PCR reaction. Primer pairs and probe sets were added corresponding to each mutation. Exemplary results are shown in Table 4. The data show that mutant DNA can be sensitively detected in wild type plasma background. 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 4 
               
               
                   
                   
               
               
                   
                   
                 Average mutant Ct 
                 Average mutant Ct 
               
               
                   
                 Mutation 
                 with mutant added 
                 in plasma only sample 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 L536Q 
                 28.62 
                 45.20 
               
               
                   
                 V422delV 
                 28.55 
                 ND 
               
               
                   
                 L536R 
                 28.55 
                 45.20 
               
               
                   
                 L536H 
                 28.40 
                 45.20 
               
               
                   
                 Y537S 
                 28.41 
                 ND 
               
               
                   
                 E380Q 
                 28.28 
                 ND 
               
               
                   
                 L536P 
                 29.41 
                 45.20 
               
               
                   
                   
               
            
           
         
       
     
     Selected Oligonucleotides 
     Allele-specific primers that show good specificity for mutant sequence were selected based on the criteria above. These are shown in Table 5 below with the primer name, and SEQ ID NO in parenthesis. Bolded entries indicate primers that show selectivity and specificity in the assay configuration in Table 6 below, though other combinations also perform well. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Selected allele-specific primers 
               
            
           
           
               
               
            
               
                 Mutation 
                 Selected allele-specific primers 
               
               
                   
               
               
                 K303R 
                 FS02 (479),  FS04 (481) , FS07 (484) 
               
               
                 S341L 
                 FS02 (239), FS03 (240), FS05 (242),  FS08 (245) , FS09 (246), FS10 (247), RS04 
               
               
                   
                 (253), RS05 (254), RS06 (255), RS07 (256), RS08 (257), RS09 (258), RS10 (259) 
               
               
                 E380Q 
                 FS01 (32), FS03 (34), FS05 (36), FS06 (37), FS07 (38), FS08 (39), FS09 (40), FS10 
               
               
                   
                 (41),  FS11 (42)   
               
               
                 V422DELV 
                 FS01 (287), FS08 (294), FS09 (295), FS10 (296), FS12, FS14 (297),  FS15 (298)   
               
               
                 L429V 
                 FS06 (66), FS08 (68), FS09 (69),  FS10 (70) , RS01 (72), RS02 (73), RS04 (75), RS05 
               
               
                   
                 (76), RS06 (77), RS07 (78), RS08 (79), RS09 (80), RS10 (81) 
               
               
                 S463P 
                   FS02 (264) , FS03 (265), FS05 (267), FS06 (268), FS07 (269), FS08 (270), FS09 
               
               
                   
                 (271), FS10 (272), RS08 (281), RS09 (282), RS10 (283), RS11 (275), RS12 
               
               
                 V533M 
                 FS01 (322), FS08 (329), FS09 (330), RS08 (345), RS09 (346), RS10 (347), RS11 
               
               
                   
                 (348), RS12 (349),  RS13 (350) , RS15 (352), RS16 (353), RS19 
               
               
                 V534E 
                 FS01 (364), FS02 (365), FS04 (367), FS05 (368), FS07 (370), FS08 (371), FS10 (373), 
               
               
                   
                 RS01 (378),  RS11 (388) , RS15 (391), RS16 (392), RS17 (393) 
               
               
                 P535H 
                 FS01 (181), FS08 (188), FS09 (189), FS10 (190), RS01 (191), RS08 (198), RS09 
               
               
                   
                 (199), RS10 (200), RS13 (203), RS14 (204), RS15 (205), RS18 (208), RS19 (209), 
               
               
                   
                 RS22 (212), RS35 (225), RS36 (226), RS38 (228), RS39 (229),  RS41 (231)   
               
               
                 L536H 
                 FS01 (96), FS06 (101), FS09 (104), FS10 (105), RS02 (107), RS05 (110), RS06 (111), 
               
               
                   
                 RS07 (112),  RS08 (113) , RS09 (114), RS10 (115), RS11 (116), RS12 (117) 
               
               
                 L536P 
                   RS04 (134) , RS05 (135), RS07 (137) 
               
               
                 L536Q 
                 FS01 (141), RS01 (151), RS04 (154), RS05 (155), RS07 (157),  RS08 (158) , RS09 
               
               
                   
                 (159), RS10 (160) 
               
               
                 L536R 
                 FS01 (161), RS02 (172), RS04 (174), RS05 (175), RS06 (176), RS07 (177),  RS08   
               
               
                   
                   (178) , RS09 (179), RS10 (180) 
               
               
                 Y537C 
                 FS01 (397), RS01 (408),  RS08 (415) , RS09 (416), RS10 (417), RS11 (418), RS12 
               
               
                   
                 (419), RS13 (420), RS19 (424) 
               
               
                 Y537N 
                 FS01 (426), RS01 (436), RS06 (441), RS10 (445), RS11 (446),  RS12 (447) , RS13 
               
               
                   
                 (448), RS14 
               
               
                 Y537S 
                 FS01 (449), RS01 (459), RS08 (466), RS09 (467), RS10 (468),  RS11 (469) , RS12 
               
               
                   
                 (470), RS13 (471), RS14 (472) 
               
               
                 D538G 
                 RS01 (14), RS08 (21), RS09 (22), RS10 (23),  RS11 (24) , RS12 (25) 
               
               
                 L536_D538 &gt; P 
                 RS01 (84), RS02 (85), RS03 (86), RS04 (87), RS05 (88), RS06 (89),  RS07 (90) , RS08 
               
               
                   
                 (91), RS09 (92), RS10 (93) 
               
               
                   
               
            
           
         
       
     
     In the case of exon 8 RS primers, any exon 8 CF primer can be used, for example, V533M_CF03 (SEQ ID NO:314) or Y537C_CF01 (SEQ ID NO:394). Any exon 8 probe can be used, for example Y537C_R_P01 (SEQ ID NO:407). In general, for any FS allele-specific primer, any corresponding CR primer and F probe can be used. Similarly for any RS allele-specific primer, any corresponding CF primer and R probe can be used. Particular examples include K303R_CR02 (SEQ ID NO:476) and K303R_F_P01 (SEQ ID NO:477); S341L_CR02 (SEQ ID NO:235) and S341L_F_P02 (SEQ ID NO:237); E380QCRP3 (SEQ ID NO:528) and E380Q_F_P01 (SEQ ID NO:31); V422delV_CR01 (SEQ ID NO:285) and V422delV_F_P01 (SEQ ID NO:286); L429V_CR01 (SEQ ID NO:59) and L429_F_P01 (SEQ ID NO:60); and S463P_CR01 (SEQ ID NO:261) and S463P_F_P01 (SEQ ID NO:262). 
     Example Assay Configuration 
     We sought to design a highly multiplexed assay to detect the maximum number of mutations in a minimal number of reaction vessels. Examples of three-vessel assays are shown below in Tables 6 and 7. Exon 8 mutations are grouped together to allow for use of a common forward primer and probe. If desired, one of skill will understand how to identify the exact exon 8 mutation(s) that results in a mutation positive signal, e.g., using sequencing or PCR with mutation-specific probes. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Exemplary assay configuration for allele-specific multiplex ESR1 mutation 
               
            
           
           
               
               
               
               
            
               
                 Reporter 
                 1 
                 2 
                 3 
               
               
                   
               
               
                 FAM 
                 L536H/L536P/L536Q/ 
                 Y537C/Y537N/Y537S/ 
                 V533M/ 
               
               
                   
                 L536R/D538G 
                 L536_D538 &gt; P 
                 V534E/ 
               
               
                   
                   
                   
                 P535H 
               
               
                 HEX 
                 S463P 
                 E380Q 
                 S341L 
               
               
                 JA270 
                 V422DELV 
                 K303R 
                 L429V 
               
               
                 CY5.5 
                 IC 
                 IC 
                 IC 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Exemplary assay configuration for allele-specific multiplex ESR1 mutation 
               
            
           
           
               
               
               
               
            
               
                 Reporter 
                 1 
                 2 
                 3 
               
               
                   
               
               
                 FAM 
                 L536H/L536P/L536Q/ 
                 Y537C/Y537N/ 
                 V533M/ 
               
               
                   
                 L536R/L536_D538 &gt; P 
                 Y537S/D538G 
                 V534E/ 
               
               
                   
                   
                   
                 P535H 
               
               
                 HEX 
                 S463P 
                 S341L 
                 E380Q 
               
               
                 JA270 
                 K303R 
                 L429V 
                 V422DELV 
               
               
                 CY5.5 
                 IC 
                 IC 
                 IC 
               
               
                   
               
            
           
         
       
     
     One of skill will understand that different configurations can be made, depending on the mutations of interest and availability of fluorescence channels. For example, the number of tubes can be increased to allow for individual detection of exon 8 mutations. As the number of fluorescence channels increases, the number of mutations that can be included in a single tube will increase. 
     Detection of E380Q mutation from FFPET Samples in Multiplex Reactions 
     DNA from 142 FFPET samples (breast, lymph node, lung, bone) from cancer patients was extracted using the cobas® DNA Sample Preparation Kit. DNA (50 ng/reaction) was analyzed for ESR1 mutations by the multiplex allele-specific PCR as described herein (performed in duplicate). Results are shown in  FIGS. 4A and 4B . One sample (IN008) was found positive for the E380Q mutation, and has a similar amplification growth curve to the E380Q mutation positive control (MC) (see  FIG. 4A ).  FIG. 4B  shows the same data without the E380Q mutation positive control. Results were confirmed using ddPCR. 
     The E380Q mutation positive sample shows a profile that is clearly distinguishable from E380 wild type and non-template control. The results also show that the multiplex assays described herein are effective for detecting specific mutations in DNA from FFPET clinical samples. 
     Detection of D538G Mutation from Plasma Samples in Multiplex Reactions 
     Clinical plasma samples (2 ml plasma) were obtained from 103 post-menopausal metastatic breast cancer patients who relapsed during treatment with an aromatase inhibitor, or within 12 months after discontinuation. DNA was extracted using the cobas® cfDNA Sample Preparation Kit and analyzed for ESR1 mutations by multiplex allele-specific PCR as described herein. Three samples tested positive for D538G, as shown in  FIG. 5 .  FIG. 5  shows results from 51 of the samples, 3 D538G positive and 48 D538 wild type. D538G mutation positive and non-template controls are also shown, and the D538G positive samples show profiles that are dearly distinguishable. Results were confirmed using ddPCR. 
     The results show that the multiplex assays described herein are effective for detecting specific mutations in DNA from non-invasive (plasma) clinical samples. 
     While the invention has been described in detail with reference to specific examples, it will be apparent to one skilled in the art that various modifications can be made within the scope of this invention. Thus the scope of the invention should not be limited by the examples described herein. All patents, publications, websites, Genbank (or other database) entries disclosed herein are incorporated by reference in their entireties.