Probe for Detection of Polymorphism in EGFR Gene, Amplification Primer, and Use Thereof

Abstract

A polymorphism-detecting probe, an amplification primer and the use thereof are provided to enable simple and highly reliable determination of different polymorphisms in an EGFR gene.

1 . A labeled probe comprising at least one oligonucleotide selected from the group consisting of oligonucleotides P1, P3, P5-P7, and P15-P18:
 (P1) An oligonucleotide comprising a sequence at least about 85% identical to a complementary nucleotide sequence of 11 to 50 nucleotides to nucleotides 251 to 261 of SEQ ID NO: 1;   (P3) An oligonucleotide comprising a sequence at least about 85% identical to a complementary nucleotide sequence of 5 to 50 nucleotides to nucleotides 257 to 261 of SEQ ID NO: 1;   (P5) An oligonucleotide comprising a sequence at least about 85% identical to a nucleotide sequence of 9 to 50 nucleotides comprising nucleotides 104 to 112 of SEQ ID NO: 2;   (P6) An oligonucleotide comprising a sequence at least about 85% identical to a nucleotide sequence of 16 to 50 nucleotides comprising nucleotides 104 to 119 of SEQ ID NO: 2;   (P7) An oligonucleotide comprising a sequence at least about 85% identical to a nucleotide sequence of 10 to 50 nucleotides comprising nucleotides 136 to 145 of SEQ ID NO: 3;   (P15) An oligonucleotide comprising a sequence at least about 85% identical to a complementary nucleotide sequence of 6 to 50 nucleotides to nucleotides 259 to 264 of SEQ ID NO: 1;   (P16) An oligonucleotide comprising a sequence at least about 85% identical to a complementary nucleotide sequence of 5 to 50 nucleotides to nucleotides 258 to 262 of SEQ ID NO: 1;   (P17) An oligonucleotide comprising a sequence at least about 85% identical to a nucleotide sequence of 16 to 50 nucleotides comprising nucleotides 249 to 264 of SEQ ID NO: 1; and   (P18) An oligonucleotide comprising a sequence at least about 85% identical to a nucleotide sequence of 8 to 50 nucleotides comprising nucleotides 257 to 264 of SEQ ID NO: 1.   
     
     
         2 . The probe according to  claim 1 , wherein
 the oligonucleotide P1 is an oligonucleotide comprising a complementary nucleotide sequence of 11 to 50 nucleotides to nucleotides 251 to 261 of SEQ ID NO: 1;   the oligonucleotide P3 is an oligonucleotide comprising a complementary nucleotide sequence of 5 to 50 nucleotides to nucleotides 257 to 261 of SEQ ID NO: 1;   the oligonucleotide P5 is an oligonucleotide comprising a nucleotide sequence of 9 to 50 nucleotides comprising nucleotides 104 to 112 of SEQ ID NO: 2;   the oligonucleotide P6 is an oligonucleotide comprising a nucleotide sequence of 16 to 50 nucleotides comprising nucleotides 104 to 119 of SEQ ID NO: 2;   the oligonucleotide P7 is an oligonucleotide comprising a nucleotide sequence of 10 to 50 nucleotides comprising nucleotides 136 to 145 of SEQ ID NO: 3;   the oligonucleotide P15 is an oligonucleotide comprising a complementary nucleotide sequence of 6 to 50 nucleotides to nucleotides 259 to 264 of SEQ ID NO: 1;   the oligonucleotide P16 is an oligonucleotide comprising a complementary nucleotide sequence of 5 to 50 nucleotides to nucleotides 258 to 262 of SEQ ID NO: 1;   the oligonucleotide P17 is an oligonucleotide comprising a nucleotide sequence of 16 to 50 nucleotides comprising nucleotides 249 to 264 of SEQ ID NO: 1; and   the oligonucleotide P18 is an oligonucleotide comprising a nucleotide sequence of 8 to 50 nucleotides comprising nucleotides 257 to 264 of SEQ ID NO: 1.   
     
     
         3 . The probe according to  claim 1 , wherein the probe is fluorescence-labeled. 
     
     
         4 . The probe according to  claim 1 , wherein
 in the oligonucleotide P1, the nucleotide corresponding to nucleotide 261 is adenine or cytosine, the nucleotide corresponding to nucleotide 251 is cytosine, and the cytosine is fluorescence-labeled;   in the oligonucleotide P3, the nucleotide corresponding to nucleotide 261 is adenine or cytosine, the nucleotide corresponding to nucleotide 257 is cytosine, and the cytosine is fluorescence-labeled;   in the oligonucleotide P5, the nucleotide corresponding to nucleotide 112 is thymine, the nucleotide corresponding to nucleotide 104 is cytosine, and the cytosine is fluorescence-labeled;   in the oligonucleotide P6, the nucleotide at position 119 is substituted by a nucleotide other than G, and in which the nucleotide corresponding to nucleotide 104 is cytosine, and the cytosine is fluorescence-labeled;   in the oligonucleotide P7, the nucleotide corresponding to nucleotide 145 is cytosine, and the cytosine is fluorescence-labeled;   in the oligonucleotide P15, the nucleotide corresponding to nucleotide 261 is adenine or cytosine, and cytosine positioned at the 3′ side relative to the nucleotide is fluorescence-labeled;   in the oligonucleotide P16, the nucleotide corresponding to nucleotide 261 is adenine or cytosine, and cytosine positioned at the 5′ side relative to the nucleotide is fluorescence-labeled;   in the oligonucleotide P17, the nucleotide corresponding to nucleotide 261 is thymine or guanine, and cytosine positioned at the 5′ side relative to the nucleotide is fluorescence-labeled; and   in the oligonucleotide P18, the nucleotide corresponding to nucleotide 261 is thymine or guanine, and cytosine positioned at the 3′ side relative to the nucleotide is fluorescence-labeled.   
     
     
         5 . The probe according to  claim 1 , wherein
 the oligonucleotide P1 includes the fluorescence-labeled nucleotide corresponding to nucleotide 251 at the 1 st  to 3 rd  position counted from the 3′ terminal;   the oligonucleotide P3 includes the fluorescence-labeled nucleotide corresponding to nucleotide 257 at the 1 st  to 3 rd  position counted from the 3′ terminal;   the oligonucleotide P5 includes the fluorescence-labeled nucleotide corresponding to nucleotide 104 at the 1 st  to 3 rd  position counted from the 5′ terminal;   the oligonucleotide P6 includes the fluorescence-labeled nucleotide corresponding to nucleotide 104 at the 1 st  to 3 rd  position counted from the 5′ terminal;   the oligonucleotide P7 includes the fluorescence-labeled nucleotide corresponding to nucleotide 145 at the 1 st  to 3 rd  position counted from the 3′ terminal;   the oligonucleotide P15 includes the fluorescence-labeled cytosine at the 1 st  to 3 rd  position counted from the 3′ terminal;   the oligonucleotide P16 includes the fluorescence-labeled cytosine at the 1 st  to 3 rd  position counted from the 5′ terminal;   the oligonucleotide P17 includes the fluorescence-labeled cytosine at the 1 st  to 3 rd  position counted from the 5′ terminal; and   the oligonucleotide P18 includes the fluorescence-labeled cytosine at the 1 st  to 3 rd  position counted from the 3′ terminal.   
     
     
         6 . The probe according to  claim 1 , wherein
 the oligonucleotide P1 includes the fluorescence-labeled nucleotide corresponding to nucleotide 251 on the 3′ terminal;   the oligonucleotide P3 includes the fluorescence-labeled nucleotide corresponding to nucleotide 257 on the 3′ terminal;   the oligonucleotide P5 includes the fluorescence-labeled nucleotide corresponding to nucleotide 104 on the 5′ terminal;   the oligonucleotide P6 includes the fluorescence-labeled nucleotide corresponding to nucleotide 104 on the 5′ terminal;   the oligonucleotide P7 includes the fluorescence-labeled nucleotide corresponding to nucleotide 145 on the 3′ terminal;   the oligonucleotide P15 includes the fluorescence-labeled cytosine on the 3′ terminal;   the oligonucleotide P16 includes the fluorescence-labeled cytosine on the 5′ terminal;   the oligonucleotide P17 includes the fluorescence-labeled cytosine on the 5′ terminal; and   the oligonucleotide P18 includes the fluorescence-labeled cytosine on the 3′ terminal.   
     
     
         7 . The probe according to  claim 3 , wherein the fluorescence-labeled oligonucleotide emits fluorescence when not hybridized to a target sequence, and fluorescent intensity decreases or increases when hybridized to the target sequence. 
     
     
         8 . The probe according to  claim 7 , wherein the fluorescence-labeled oligonucleotide emits fluorescence when not hybridized to the target sequence, and the fluorescent intensity decreases when hybridized to the target sequence. 
     
     
         9 . The probe according to  claim 1 , wherein the nucleotide length of the oligonucleotide according to P1 is 11 to 30 nucleotides, the nucleotide length of each of the oligonucleotides according to P3 and P5 to P7 is 10 to 30 nucleotides, the nucleotide length of the oligonucleotide according to P15 is 16 to 30 nucleotides, the nucleotide length of the oligonucleotide according to P16 is 18 to 30 nucleotides, the nucleotide length of the oligonucleotide according to P17 is 20 to 30 nucleotides, and the nucleotide length of the oligonucleotide according to P18 is 20 to 30 nucleotides. 
     
     
         10 . The probe according to  claim 1 , wherein the probe is configured for use in melting curve analysis. 
     
     
         11 . A method of detecting a polymorphism in an EGFR gene in a sample using the probe according to  claim 1 , comprising
 adding to the sample the probe according to  claim 1 ,   obtaining a melting curve for the probe, and   determining the melting temperature of the probe from the melting curve,   wherein the melting temperature indicates the presence of the polymorphism in the EGFR gene.   
     
     
         12 . The method according to  claim 11 , wherein the method detects an EGFR exon20 T790M polymorphism. 
     
     
         13 . A method of determining efficacy of EGFR-TKI or a resistance to EGFR-TKI comprising steps of:
 detecting a polymorphism in an EGFR gene using the method according to  claim 10 ; and   determining efficacy of or a resistance to EGFR-TKI based on the presence or the absence of the polymorphism.   
     
     
         14 . A reagent kit for detecting a polymorphism in an EGFR gene, wherein the kit includes the probe according to  claim 1 . 
     
     
         15 . The kit according to  claim 14 , wherein the kit further includes a probe for detecting an EGFR exon20 T790M polymorphism. 
     
     
         16 . The kit according to  claim 14  including a primer for amplifying a domain including a sequence hybridizing with at least one of the oligonucleotides according to P1, P3, and P15-P18 in the nucleotide sequence of SEQ ID NO: 1 of the EGFR gene, a domain including a sequence hybridizing with at least one of the oligonucleotides according to P5 and P6 in the nucleotide sequence of SEQ ID NO: 2, or a domain including a sequence hybridizing with the oligonucleotide according to P7 in the nucleotide sequence of SEQ ID NO: 3. 
     
     
         17 . The kit according to  claim 16 , wherein the kit further includes a probe for detecting an EGFR exon20 T790M polymorphism. 
     
     
         18 . A primer selected from the group consisting of oligonucleotides P8 to P13:
 (P8) An oligonucleotide of 10 to 50 nucleotides homologous to nucleotides 224 to 233 of SEQ ID NO: 1, and in which the 3′ terminal is the nucleotide C at position 233.   (P9) An oligonucleotide of 10 to 50 nucleotides complementary to nucleotides 284 to 293 of SEQ ID NO: 1, and in which the 3′ terminal is the nucleotide C complementary to the nucleotide G at position 284.   (P10) An oligonucleotide of 10 to 50 nucleotides complementary to nucleotides 290 to 299 of SEQ ID NO: 1, and in which the 3′ terminal is the nucleotide C complementary to the nucleotide G at position 290.   (P11) An oligonucleotide of 10 to 50 nucleotides homologous to nucleotides 86 to 95 of SEQ ID NO: 2, and in which the 3′ terminal is the nucleotide G at position 95.   (P12) An oligonucleotide of 10 to 50 nucleotides homologous to nucleotides 64 to 73 of SEQ ID NO: 2, and in which the 3′ terminal is the nucleotide C at position 73.   (P13) An oligonucleotide of 10 to 50 nucleotides complementary to nucleotides 155 to 164 of SEQ ID NO: 2, and in which the 3′ terminal is the nucleotide C complementary to the nucleotide G at position 155.