Polynucleotide primers

Abstract

A polynucleotide primer comprising at least the final six nucleotides of one of the following primer sequences, or a sequence complementary thereto: SEQ. ID NOS. 1 to 18, 21 to 45 or 74 to 77.

1 . A polynucleotide comprising at least the final six nucleotides of one of the following primer sequences, or a sequence complementary thereto: SEQ. ID NOS. 1 to 18, 21 to 45 or 74 to 77. 
     
     
         2 . A polynucleotide according to  claim 1  wherein the polynucleotide is less than 100 nucleotides long, preferably less than 80 nucleotides long, more preferably less than 60 nucleotides long, more preferably less than 40 nucleotides, more preferably less than 30 nucleotides long. 
     
     
         3 . A polynucleotide according to  claim 1  comprising at least 75% of the final 8, 10, 12, 14, 16, 17, 18 or 20 nucleotides, or the entirety of one of the following primer sequences, or a sequence complementary thereto: SEQ. ID NOS. 1 to 18, 21 to 45 or 74 to 77. 
     
     
         4 . A polynucleotide according to  claim 1  further comprising a quencher group and a fluorophore group. 
     
     
         5 . A polynucleotide according to  claim 4  wherein the quencher group and the fluorophore group are separated by a nucleotide tail sequence comprising first and second regions, the nucleotides of the first region being complementary to but in reverse order from the nucleotides of the second region, such that hybridisation of the first region to the second group results in the quencher group to be sufficiently close to the fluorophore group to quench the fluorophore group. 
     
     
         6 . A polynucleotide according to  claim 5  wherein the tail sequence further comprises a third region having a sequence complementary to a region of the EGFR gene. 
     
     
         7 . A polynucleotide according to  claim 6  comprising at least the final six nucleotides of SEQ. ID NO. 3 or 10 and the tail sequence comprises SEQ. ID NO. 19. 
     
     
         8 . A polynucleotide according to  claim 6  comprising at least the final six nucleotides of SEQ. ID NOS. 6 or 12 and the tail sequence comprises SEQ. ID NO. 20. 
     
     
         9 . A polynucleotide according to  claim 6  comprising at least the final six nucleotides of SEQ. ID NO. 76 and the tail sequence comprises SEQ. ID NO. 77. 
     
     
         10 . A polynucleotide according to  claim 4  wherein the quencher group comprises black hole quencher 1 (BHQ1) and the fluorophore group comprises FAM. 
     
     
         11 . A polynucleotide according to  claim 4  wherein the quencher group comprises black hole quencher 2 (BHQ2) and the fluorophore comprises Cal Red. 
     
     
         12 . A kit comprising at least a pair of polynucleotides wherein the pair of polynucleotides comprises at least four or five of the final six nucleotides of one of the following pairs of primer sequences, respectively, or sequences complementary thereto: SEQ. ID NO. 1 and SEQ. ID NO. 15, or SEQ. ID NO. 2 and SEQ. ID NO 15, or SEQ. ID NO. 3 and SEQ. ID NO. 15, or SEQ. ID NO. 4 and SEQ. ID NO. 15, or SEQ. ID NO. 5 and SEQ. ID NO. 15, or SEQ. ID NO. 6 and SEQ. ID NO. 16, or SEQ. ID NO. 7 and SEQ. ID NO. 17, or SEQ. ID NO. 8 and SEQ. ID NO. 18, or SEQ. ID NO. 9 and SEQ. ID NO. 18, or SEQ. ID NO. 10 and SEQ. ID NO. 15, or SEQ. ID NO. 11 and SEQ. ID NO. 15 or SEQ. ID NO. 12 and SEQ. ID NO. 16, or SEQ. ID NO. 13 and SEQ. ID NO. 17, or SEQ. ID NO. 14 and SEQ. ID NO. 18 or SEQ. ID NO. 21 and SEQ. ID NO. 15, or SEQ. ID NO. 22 and SEQ. ID NO. 24, or SEQ. ID NO. 23 and SEQ. ID NO. 24, or SEQ. ID NO. 25 and SEQ. ID NO. 41, or SEQ. ID NO. 26 and SEQ. ID NO. 41, or SEQ. ID NO. 27 and SEQ. ID NO. 41, or SEQ. ID NO. 28 and SEQ. ID NO. 41, or SEQ. ID NO. 29 and SEQ. ID NO. 42, or SEQ. ID NO. 30 and SEQ. ID NO. 43, or SEQ. ID NO. 31 and SEQ. ID NO. 44, or SEQ. ID NO. 32 and SEQ. ID NO. 44, or SEQ. ID NO. 33 and SEQ. ID NO. 41, or SEQ. ID NO. 34 and SEQ. ID NO. 45, or SEQ. ID NO. 35 and SEQ. ID NO. 41, or SEQ. ID NO. 36 and SEQ. ID NO. 41, or SEQ. ID NO. 37 and SEQ. ID NO. 42, or SEQ. ID NO. 38 and SEQ. ID NO. 43, or SEQ. ID NO. 39 and SEQ. ID NO. 44, or SEQ. ID NO. 40 and SEQ. ID NO. 45, or SEQ. ID NO. 74 and SEQ. ID NO. 76 or SEQ. ID NO. 75 and SEQ. ID NO. 76. 
     
     
         13 . A kit according to  claim 12  wherein the pair of polynucleotides are each polynucleotides comprising at least the final six nucleotides of one of the following primer sequences, or a sequence complementary thereto: SEQ. ID NOS. 1 to 18, 21 to 45 or 74 to 77. 
     
     
         14 . A kit comprising at least a set of three polynucleotides, wherein the set of three polynucleotides comprises at least four or five of the final six nucleotides of one of the following sets of three primer sequences, respectively, or sequences complementary thereto: SEQ. ID NOS. 1, 10 and 15, or SEQ. ID NOS. 2, 10 and 15, or SEQ. ID NOS. 3, 10 and 15, or SEQ. ID NOS. 4, 11 and 15, or SEQ. ID NOS. 5, 11 and 15, or SEQ. ID NOS. 6, 12 and 16, or SEQ. ID NOS. 7, 13 and 17, or SEQ. ID NOS. 8, 14 and 18, or SEQ. ID NOS. 9, 14 and 18 or SEQ. ID NOS. 21, 10 and 15, or SEQ. ID NOS. 22, 23 and 24, or SEQ. ID NOS. 25, 35 and 41, or SEQ. ID NOS. 26, 35 and 41, or SEQ. ID NOS. 27, 36 and 41, or SEQ. ID NOS. 28, 36 and 41, or SEQ. ID NOS. 29, 32 and 42, or SEQ. ID NOS. 30, 38 and 43, or SEQ. ID NOS. 31, 39 and 44, or SEQ. ID NOS. 32, 39 and 44, or SEQ. ID NOS. 33, 35 and 41, or SEQ. ID NOS. 34, 40 and 45, SEQ. ID NOS. 74, 75 and 76. 
     
     
         15 . A kit according to  claim 14  wherein the set of three polynucleotides are each polynucleotides comprising at least the final six nucleotides of one of the following primer sequences, or a sequence complementary thereto: SEQ. ID NOS. 1 to 18, 21 to 45 or 74 to 77. 
     
     
         16 . A kit according to  claim 12  further comprising nucleotide triphosphates, a polymerisation enzyme and/or a buffer solution. 
     
     
         17 . A method of detecting the presence or absence of a mutation in the EGFR gene comprising the steps of:
 a) mixing a nucleic acid sample comprising at least a fragment of the EGFR gene with a polynucleotide complementary to a region of the fragment of the EGFR gene; and   b) detecting hybridisation of the polynucleotide to the nucleic acid sample wherein hybridisation indicates the presence or absence of a mutation.   
     
     
         18 . A method according to  claim 17  wherein the polynucleotide is a polynucleotide comprising at least the final six nucleotides of one of the following primer sequences, or a sequence complementary thereto: SEQ. ID NOS. 1 to 18, 21 to 45 or 74 to 77 and comprises at least four or five of the final six nucleotides of SEQ. ID NOS. 1 to 9, 21, 22, 25 to 34 or 75 or sequences complementary thereto and step b) indicates the presence of a mutation. 
     
     
         19 . A method according to  claim 17  wherein the polynucleotide is a polynucleotide comprising at least the final six nucleotides of one of the following primer sequences, or a sequence complementary thereto: SEQ. ID NOS. 1 to 18, 21 to 45 or 74 to 77 and comprises at least four or five of the final six nucleotides of SEQ. ID NOS. 10 to 14, 23, 35 to 40 or 74 or sequences complementary thereto; and step b) indicates the absence of a mutation. 
     
     
         20 . A method according to  claim 17  further comprising the step of, prior to step a), amplifying the number of copies of the fragment of the EGFR gene using thermal cycling nucleic acid amplification, preferably PCR. 
     
     
         21 . A method according to  claim 17 , wherein step b) comprises carrying out DNA polymerisation using the polynucleotide as a first primer and detecting the extension product of polymerisation. 
     
     
         22 . A method according to  claim 20  wherein step b) comprises the step of mixing the nucleic acid sample and the polynucleotide with a second primer which corresponds to a region of the fragment of the EGFR sequence downstream of the region to which the polynucleotide is complementary and carrying out PCR on the mixture. 
     
     
         23 . A method according to  claim 22  wherein the second primer comprises: SEQ. ID NO. 15 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NOS. 1 to 5, 10, 11 or 21; SEQ. ID NO. 16 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NOS. 6 or 12; SEQ. ID NO. 17 and the polynucleotide comprises at least for or five of the final six nucleotides of SEQ. ID NOS. 7 or 13; SEQ. ID NO. 18 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NOS. 8, 9 or 14; SEQ. ID NO. 24 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NOS: 22 or 23; SEQ. ID NO. 41 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NOS. 25 to 28, 33, 35 or 36; SEQ. ID NO. 42 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NOS. 29 or 37; SEQ. ID NO. 43 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NOS. 30 to 38; SEQ. ID NO. 44 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NOS. 31, 32 or 39; SEQ. ID NO. 45 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NOS. 34 or 40; or SEQ. ID NO. 76 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NO. 74 or 75. 
     
     
         24 . A method according to  claim 21  wherein step b) comprises the step of mixing the nucleic acid sample and the polynucleotide with a second primer which corresponds to a region of the fragment of the EGFR sequence downstream of the region to which the polynucleotide is complementary and carrying out PCR on the mixture. 
     
     
         25 . A method according to  claim 24  wherein the second primer comprises: SEQ. ID NO. 15 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NOS. 1 to 5, 10, 11 or 21; SEQ. ID NO. 16 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NOS. 6 or 12; SEQ. ID NO. 17 and the polynucleotide comprises at least for or five of the final six nucleotides of SEQ. ID NOS. 7 or 13; SEQ. ID NO. 18 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NOS. 8, 9 or 14; SEQ. ID NO. 24 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NOS: 22 or 23; SEQ. ID NO. 41 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NOS. 25 to 28, 33, 35 or 36; SEQ. ID NO. 42 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NOS. 29 or 37; SEQ. ID NO. 43 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NOS. 30 to 38; SEQ. ID NO. 44 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NOS. 31, 32 or 39; SEQ. ID NO. 45 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NOS. 34 or 40; or SEQ. ID NO. 76 and the polynucleotide comprises at least four or five of the final six nucleotides of SEQ. ID NO. 74 or 75. 
     
     
         26 . A method according to  claim 21  wherein step a) comprises the step of mixing the nucleic acid sample with a pair of a mutation specific polynucleotide and a wild-type specific polynucleotide, the pair being selected from at least four or five of the final six sequences of: SEQ. ID NO: 1 and SEQ. ID NO: 10; SEQ. ID NO: 2 and SEQ. ID NO: 10; SEQ. ID NO: 3 and SEQ. ID NO: 10; SEQ. ID NO: 4 and SEQ. ID NO: 11; SEQ. ID NO: 5 and SEQ. ID NO: 11; SEQ. ID NO: 6 and SEQ. ID NO: 12; SEQ. ID NO: 7 and SEQ. ID NO: 13; SEQ. ID NO: 8 and SEQ. ID NO: 14; SEQ. ID NO: 9 and SEQ. ID NO: 14; SEQ. ID NO: 21 and SEQ. ID NO: 10; SEQ. ID NO: 22 and SEQ. ID NO: 23; SEQ. ID NO: 25 and SEQ. ID NO: 35; SEQ. ID NO: 26 and SEQ. ID NO: 35; SEQ. ID NO: 27 and SEQ. ID NO: 36; SEQ. ID NO: 28 and SEQ. ID NO: 36; SEQ. ID NO: 29 and SEQ. ID NO: 37; SEQ. ID NO: 30 and SEQ. ID NO: 38; SEQ. ID NO: 31 and SEQ. ID NO: 39; SEQ. ID NO: 32 and SEQ. ID NO: 39; SEQ. ID NO: 33 and SEQ. ID NO: 35; SEQ. ID NO: 34 and SEQ. ID NO: 40; or SEQ. ID NO. 74 and SEQ. ID NO. 75. 
     
     
         27 . A method according to  claim 26  wherein the nucleic acid sample comprises wild-type sequences and mutated sequences and further comprising step c) wherein the number of amplification cycles required to amplify the wild-type sequences to a predetermined quantity is compared with the number of amplification cycles required to amplify the mutated sequences to the predetermined quantity thereby providing an indication of the ratio of the wild type sequences to mutated sequences in the sample. 
     
     
         28 . A method according to  claim 27  wherein the nucleic acid sample comprises a portion of tumourous tissue and a portion of non-tumourous tissue and wherein step c) further comprises the step determining the ratio of tumourous tissue to non-tumourous tissue in the sample. 
     
     
         29 . A method according to  claim 27  further comprising the step of, prior to step a), enriching the nucleic acid sample to increase the ratio of tumourous tissue to non-tumourous tissue in the sample. 
     
     
         30 . A method according to  claim 28  further comprising the step of, prior to step a), enriching the nucleic acid sample to increase the ratio of tumourous tissue to non-tumourous tissue in the sample. 
     
     
         31 . A method according to  claim 22  wherein step b) comprises detecting if amplification of at least a portion of the EGFR gene occurs. 
     
     
         32 . A method according to  claim 17  wherein the polynucleotide comprise a quencher group and a fluorophore group and wherein step b) comprises exposing the mixture to light of a wavelength to which the fluorophore is responsive in the absence of the quencher group and detecting light at the wavelength emitted by the fluorophore group in the absence of the quencher group.