Claims for Patent: 5,550,020

Summary for Patent: 5,550,020

Title:	Method, reagents and kit for diagnosis and targeted screening for retinoblastoma
Abstract:	Reliable and cost effective testing for mutations in the RB1 gene can be accomplished by quantitatively amplifying exons of the sample RB1 gene using primers complementary to intron regions flanking each exon; and then determining the lengths and/or quantities of the amplification products for each exon and comparing that length or quantity to the length or quantity of amplification products obtained when a wild-type RB1 gene is amplified using the same primers. Differences in length between an amplified sample exon and the corresponding amplified wild-type exon reflect the occurrence of an insertion or deletion mutation in the sample RB1 gene. Differences in quantity reflect the complete absence of an exon, or heterozygosity for a mutant exon. Next, the nucleic acid sequence of each exon found to contain an insertion or deletion mutation is determined, or of all exons in the event no insertion or deletion mutations are identified. Preferably, the amplification of the exons is multiplexed so that more than one exon is amplified in a single vessel using sets of primers which provide gene fragments of distinctive lengths when used to amplify a normal RB1 gene.
Inventor(s):	Gallie; Brenda L. (Toronto, CA), Dunn; James M. (Scarborough, CA), Stevens; John K. (Mississauga, CA)
Assignee:	Visible Genetics Inc. (Toronto, CA) HSC Research & Development (Toronto, CA)
Application Number:	08/271,942
Patent Claims:	1. A method for identifying mutations in a sample retinoblastoma gene comprising the steps of: (a) quantitatively amplifying one or more exons of the sample retinoblastoma gene using primers complementary to intron regions immediately flanking each amplified exon; (b) determining the lengths of the amplification products for each amplified sample exon and comparing that length to the length of amplification products obtained when a wild-type retinoblastoma gene is amplified using the same primers, whereby differences in length between an amplified sample exon and the corresponding amplified wild-type exon reflect the occurrence on an insertion or deletion mutation in the sample retinoblastoma gene; and (c) determining the nucleic acid sequence of each exon identified in step (b) to contain an insertion or deletion mutation, or in the event no insertion or deletion mutations are identified, the nucleic acid sequence of at least one exon of the retinoblastoma gene until all exons have been sequenced or until a mutation has been detected. 2. The method according to claim 1, further comprising the step of determining the quantity of the amplification products and comparing this quantity to that of the corresponding amplified wild-type exon. 3. The method according to claim 1, wherein at least one exon selected from consisting of exons 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24 is sequenced. 4. The method according to claim 1, wherein the lengths of the amplification products for each amplified sample exon are determined by electrophoresis on a gel having a resolution of one-base. 5. The method according to claim 1, wherein the lengths of the amplification products for each amplified sample exon are determined by electrophoresis on a polyacrylamide gel having a resolution of one-base. 6. The method according to claim 1, wherein at least two of the exons of the sample RB1 gene are coamplifed in a single reaction. 7. The method according to claim 1, wherein at least three of the exons of the sample RB1 gene are coamplifed in a single reaction. 8. The method according to claim 7, wherein the primers for the coamplified exons of the RB1 gene are selected to provide amplified fragments of different lengths. 9. The method according to claim 8, wherein lengths of the amplified fragments differ by at least 5 bases. 10. The method according to claim 1, wherein at least exon 1 of the RB1 gene is quantitatively amplified, and wherein the primers used are GCCCCAGTTC CCCACAGAC [Seq ID No.: 13] and ACCCCTCGCC CAAGAACCCA [Seq ID No.: 14]. 11. The method according to claim 1, wherein at least exon 2 of the RB1 gene is quantitatively amplified, and wherein the primers used are ACTGTGTGGT ATCCTTATTT TG [Seq ID No.: 1] and ATAGTGATTT GAAGTTGGTT TTA [Seq ID No.: 2]. 12. The method according to claim 1, wherein at least exon 3 of the RB1 gene is quantitatively amplified, and wherein the primers used are ATACAGTTTT AACATAGTAT CCA [Seq ID No.: 3] and AAGTCTATTG AGAGGAAAAT CC [Seq ID No.: 4]. 13. The method according to claim 1, wherein at least exon 4 of the RB1 gene is quantitatively amplified, and wherein the primers used are TTGAAAACGA AATAACAC [Seq ID No.: 39] and ATAAAAAATC AGAGTGTAAC CC [Seq ID No.: 40]. 14. The method according to claim 1, wherein at least exon 5 of the RB1 gene is quantitatively amplified, and wherein the primers used are CTACTATGAC TTCTAAATTA CG [Seq ID No.: 5] and TCAAGATGTT TGAGATTATT CC [Seq ID No.: 6]. 15. The method according to claim 1, wherein at least exon 6 of the RB1 gene is quantitatively amplified, and wherein the primers used are AAAGAAACAC CCAAAAGATA [Seq ID No.: 25] and TAATAAGCCA AGCAGAGAAT GA [Seq ID No.: 26]. 16. The method according to claim 1, wherein at least exon 7 of the RB1 gene is quantitatively amplified, and wherein the primers used are TTATGGATAT ACTCTACCCT GC [Seq ID No.: 27] and CCTCCATTTG TTGTATTTTG AC [Seq ID No.: 28]. 17. The method according to claim 1 wherein at least exon 8 of the RB1 gene is quantitatively amplified, and wherein the primers used are TCTAATGAAA CCTAATAAGT A [Seq ID No.: 15] and TGCTCATAAC AAAAGAAGTA A [Seq ID No.: 16]. 18. The method according to claim 1, wherein at least exon 9 of the RB1 gene is quantitatively amplified, and wherein the primers used are TCAAGAGTCA AGAGATTAGA [Seq ID No.: 29] and ATTATCCTCC CTCCACAGTC TC [Seq ID No.: 30]. 19. The method according to claim 1, wherein at least exon 10 of the RB1 gene is quantitatively amplified, and wherein the primers used are GTGCTGAGAG ATGTAATGA [Seq ID No.: 31] and TATCTAAAGG TCACTAAGC [Seq ID No.: 32]. 20. The method according to claim 1, wherein at least exon 11 of the RB1 gene is quantitatively amplified, and wherein the primers used are TGAGACAACA GAAGCATTAT [Seq ID No.: 33] and TGAACAAATC TGAAACACTAT [Seq ID No.: 34 ]. 21. The method according to claim 1, wherein at least exon 12 of the RB1 gene is quantitatively amplified, and wherein the primers used are CTCCCTTCAT TGCTTAACAC AT [Seq ID No.: 35] and AAAAGCAAGA AAAGATTATG G [Seq ID No.: 36]. 22. The method according to claim 1, wherein at least exon 13 of the RB1 gene is quantitatively amplified, and wherein the primers used are TGCTTATGTT CAGTAGTTGT G [Seq ID No.: 7] and TAATGGGGTG GGAGGTAGTT T [Seq ID No.: 8]. 23. The method according to claim 1, wherein at least exon 14 of the RB1 gene is quantitatively amplified, and wherein the primers used are GTGATTTTCT AAAATAGCAG GC [Seq ID No.: 41 ] and CCAGGATGAT CTTGATGCC [Seq ID No.: 42]. 24. The method according to claim 1, wherein at least exons 15 and 16 of the RB1 gene are quantitatively amplified using a single pair of primers, and wherein the primers used are CAATGCTGAC ACAAATAAGG TT [Seq ID No.: 49] and CCCCCGACCA AAGAAACACA [Seq ID No.: 50]. 25. The method according to claim 1, wherein at least exon 17 of the RB1 gene is quantitatively amplified, and wherein the primers used are ACCTTTCTAC TGTTTTCTTT GT [Seq ID No.: 51] and AAACACCTCT CACTAACAAT [Seq ID No.: 52]. 26. The method according to claim 1, wherein at least exon 18 of the RB1 gene is quantitatively amplified, and wherein the primers used are TTTTTGTGTG TGGGAAGTAC A [Seq ID No.: 17] and ATTCTATTCC CTACAGTTTC TT [Seq ID No.: 18]. 27. The method according to claim 1, wherein at least exon 19 of the RB1 gene is quantitatively amplified, and wherein the primers used are TGTATAATCT GTGATTCTTA GC [Seq ID No.: 53] and GCAACATTAT CATTTCCATT TT [Seq ID No.: 54]. 28. The method according to claim 1, wherein at least exon 20 of the RB1 gene is quantitatively amplified, and wherein the primers used are GAAAAGAGTG GTAGAAAAGA GG [Seq ID No.: 43] and TAACAAGTAA GTAGGGAGGA GA [Seq ID No.: 44]. 29. The method according to claim 1, wherein at least exon 21 of the RB1 gene is quantitatively amplified, and wherein the primers used are GGCTAAAAGA AAGAAAATGG [Seq ID No.: 19] and TTACCTATGT TATGTTATGG [Seq ID No.: 20]. 30. The method according to claim 1, wherein at least exon 22 of the RB1 gene is quantitatively amplified, and wherein the primers used are TATGTGCTTC TTACCAGTCA AA [Seq ID No.: 21] and GGAGTCATTT TTGTTGGTGT TG [Seq ID No.: 22]. 31. The method according to claim 1, wherein at least exon 23 of the RB1 gene is quantitatively amplified, and wherein the primers used are AATCTAATGT AATGGGTCCA CC [Seq ID No.: 23] and ATCAAAATAA TCCCCCTCTC AT [Seq ID No.: 24]. 32. The method according to claim 1, wherein at least exon 24 of the RB1 gene is quantitatively amplified, and wherein the primers used are GTATTTATGC TCATCTCTGC [Seq ID No.: 45] and GTGTTTGAAT AACTGCATTT GG [Seq ID No.: 46]. 33. The method according to claim 1, wherein at least exon 25 of the RB1 gene is quantitatively amplified, and wherein the primers used are TCAAACTATA ACTTGAGGTT GC [Seq ID No.: 9] and AAAGAAATTG GTATAAGCCA GG [Seq ID No.: 10]. 34. The method according to claim 1, wherein at least exon 26 of the RB1 gene is quantitatively amplified, and wherein the primers used are CGAAAGCATC ATAGTTACTG G [Seq ID No.: 47] and ATATAACGAA AAGACTTCTT GC [Seq ID No.: 48]. 35. The method according to claim 1, wherein at least exon 27 of the RB1 gene is quantitatively amplified, and wherein the primers used are ACTTACCCAG TACCATCAAT GC [Seq ID No.: 37] and TCAAGTGGCT TAGGAATCAC CC [Seq ID No.: 38]. 36. The method according to claim 1, wherein exons 2, 3, 5, 13 and 25 of the sample RB1 gene are coamplifed in a single reaction. 37. The method according to claim 36, wherein the primers employed for the coamplification are as follows: for exon 2, Seq.ID No.: 1 and Seq.ID No.: 2; for exon 3, Seq.ID No.: 3 and Seq.ID No.: 4; for exon 5, Seq.ID No.: 5 and Seq.ID No.: 6; for exon 13, Seq.ID No.: 7 and Seq.ID No.: 8; and for exon 25, Seq.ID No.: 9 and Seq.ID No.: 10. 38. The method according to claim 1, wherein exons 1, 8, 18, 21, 22, and 23 of the sample RB1 gene are coamplified in a single reaction. 39. The method according to claim 38, wherein the primers employed for the coamplification are as follows: for exon 1, Seq.ID No.: 13 and Seq.ID No.: 14; for exon 8, Seq.ID No.: 15 and Seq.ID No.: 16; for exon 18, Seq.ID No.: 17 and Seq.ID No.: 18; for exon 21, Seq.ID No.: 19 and Seq.ID No.: 20; for exon 22, Seq.ID No.: 21 and Seq.ID No.: 22; and for exon 23, Seq.ID No.: 23 and Seq.ID No.: 24. 40. The method according to claim 1, wherein exons 6, 7, 8, 9, 10, 11, 12, and 27 of the sample RB1 gene are coamplified in a single reaction. 41. The method according to claim 40, wherein the primers employed for the coamplification are as follows: for exon 6, Seq.ID No.: 25 and Seq.ID No.: 26; for exon 7, Seq.ID No.: 27 and Seq.ID No.: 28; for exon 9, Seq.ID No.: 29 and Seq.ID No.: 30; for exon 10, Seq.ID No.: 31 and Seq.ID No.: 32; for exon 11, Seq.ID No.: 33 and Seq.ID No.: 34; for exon 12, Seq.ID No.: 35 and Seq.ID No.: 36; and for exon 27, Seq.ID No: 37 and Seq.ID No.: 38. 42. The method according to claim 1, wherein exons 4, 14, 20, 24 and 26 of the sample RB1 gene are coamplified in a single reaction. 43. The method according to claim 42, wherein the primers employed for the coamplification are as follows: for exon 4, Seq.ID No.: 39 and Seq.ID No.: 40; for exon 14, Seq.ID No.: 41 and Seq.ID No.: 42; for exon 20, Seq.ID No.: 43 and Seq.ID No.: 44; for exon 24, Seq.ID No.: 45 and Seq.ID No.: 46; and for exon 26, Seq.ID No.: 47 and Seq.ID No.: 48. 44. The method according to claim 1, wherein exons 15, 16, 17, and 19 of the sample RB1 gene are coamplified in a single reaction. 45. The method according to claim 44, wherein the primers employed for the coamplification are as follows: for exons 15 and 16, Seq.ID No.: 49 and Seq.ID No.: 50; for exon 17, Seq.ID No.: 51 and Seq.ID No.: 52; and for exon 19, Seq.ID No.: 53 and Seq.ID NO.: 54. 46. The method for genetic screening of family members of an individual diagnosed as having retinoblastoma, comprising the steps of: (a) obtaining a patient blood sample from the diagnosed individual; (b) quantitatively amplifying at least one exon of the retinoblastoma gene in cells from the patient blood sample using primers complementary to intron regions immediately flanking each exon amplified; (c) determining the length of the amplification product for each exon amplified and comparing that length to the length of amplification products obtained when a wild-type retinoblastoma gene is amplified using the same primers, whereby differences in length between an amplified sample exon and the corresponding amplified wild-type exon reflect the occurrence of an inherited insertion or deletion mutation in the retinoblastoma gene of the diagnosed individual; and (d) if an inherited mutation is identified, obtaining blood samples from the biological parents of the diagnosed individual; quantitatively amplifying the exon of the retinoblastoma gene found to contain an insertion or deletion mutation in the patient blood sample in cells from the parent blood samples using the same primers used to amplify the exons in the patient blood sample; and determining the length of the amplification product for the exon in the amplified parent blood samples and comparing that length to the length of amplification products obtained when the patient blood sample was amplified. 47. The method according to claim 46, further comprising the step of determining the quantity of the amplification products and comparing this quantity to that of the corresponding amplified wild-type exon. 48. The method according to claim 46, further comprising the step of determining the sequence of the exon containing the inherited mutation. 49. The method according to claim 46, wherein the length of the amplified fragments is determined by electrophoresis on a gel having a resolution capable of detecting length differences of one base pair. 50. The method according to claim 46, wherein the length of the amplified fragments is determined by electrophoresis on a polyacrylamide gel having a resolution capable of detecting length differences of one base pair. 51. The method according to claim 46, wherein the exons of the RB1 gene in cells from the patient blood sample are coamplifed in groups of three or more exons per amplification reaction. 52. The method according to claim 46, wherein at least one parent is found to carry the mutation found in the patient, further comprising the steps of obtaining aunt/uncle blood samples from the siblings of the parent found to carry the mutation; quantitatively amplifying the exon of the RB1 gene found to contain an insertion or deletion mutation in the patient blood sample in cells from the aunt/uncle blood samples using the same primers used to amplify the exons in the patient blood sample; and determining the length of the amplification product for the exon in the amplified aunt/uncle blood samples and comparing that length to the length of amplification products obtained when the patient blood sample was amplified. 53. The method according to claim 46, wherein at least exon 1 of the RB1 gene is quantitatively amplified, and wherein the primers used are GCCCCAGTTC CCCACAGAC [Seq ID No.: 13] and ACCCCTCGCC CAAGAACCCA [Seq ID No.: 14]. 54. The method according to claim 46, wherein at least exon 2 of the RB1 gene is quantitatively amplified, and wherein the primers used are ACTGTGTGGT ATCCTTATTT TG [Seq ID No.: 1] and ATAGTGATTT GAAGTTGGTT TTA [Seq ID No.: 2]. 55. The method according to claim 46, wherein at least exon 3 of the RB1 gene is quantitatively amplified, and wherein the primers used are ATACAGTTTT AACATAGTAT CCA [Seq ID No.: 3] and AAGTCTATTG AGAGGAAAAT CC [Seq ID No.: 4]. 56. The method according to claim 46, wherein at least exon 4 of the RB1 gene is quantitatively amplified, and wherein the primers used are TTGAAAACGA AATAACAC [Seq ID No.: 39] and ATAAAAAATC AGAGTGTAAC CC [Seq ID No.: 40]. 57. The method according to claim 46, wherein at least exon 5 of the RB1 gene is quantitatively amplified, and wherein the primers used are CTACTATGAC TTCTAAATTA CG [Seq ID No.: 5] and TCAAGATGTT TGAGATTATT CC [Seq ID No.: 6]. 58. The method according to claim 46, wherein at least exon 6 of the RB1 gene is quantitatively amplified, and wherein the primers used are AAAGAAACAC CCAAAAGATA [Seq ID No.: 25] and TAATAAGCCA AGCAGAGAAT GA [Seq ID No.: 26]. 59. The method according to claim 46, wherein at least exon 7 of the RB1 gene is quantitatively amplified, and wherein the primers used are TTATGGATAT ACTCTACCCT GC [Seq ID No.: 27] and CCTCCATTTG TTGTATTTTG AC [Seq ID No.: 28]. 60. The method according to claim 46, wherein at least exon 8 of the RB1 gene is quantitatively amplified, and wherein the primers used are TCTAATGAAA CCTAATAAGT A [Seq ID No.: 15] and TGCTCATAAC AAAAGAAGTA A [Seq ID No.: 16]. 61. The method according to claim 46, wherein at least exon 9 of the RB1 gene is quantitatively amplified, and wherein the primers used are TCAAGAGTCA AGAGATTAGA [Seq ID No.: 29] and ATTATCCTCC CTCCACAGTC TC [Seq ID No.: 30]. 62. The method according to claim 46, wherein at least exon 10 of the RB1 gene is quantitatively amplified, and wherein the primers used are GTGCTGAGAG ATGTAATGA [Seq ID No.: 31] and TATCTAAAGG TCACTAAGC [Seq ID No.: 32]. 63. The method according to claim 46, wherein at least exon 11 of the RB1 gene is quantitatively amplified, and wherein the primers used are TGAGACAACA GAAGCATTAT [Seq ID No.: 33] and TGAACAAATC TGAAACACTAT [Seq ID No.: 34]. 64. The method according to claim 46, wherein at least exon 12 of the RB1 gene is quantitatively amplified, and wherein the primers used are CTCCCTTCAT TGCTTAACAC AT [Seq ID No.: 35] and AAAAGCAAGA AAAGATTATG G [Seq ID No.: 36]. 65. The method according to claim 46, wherein at least exon 13 of the RB1 gene is quantitatively amplified, and wherein the primers used are TGCTTATGTT CAGTAGTTGT G [Seq ID No.: 7] and TAATGGGGTG GGAGGTAGTT T [Seq ID No.: 8]. 66. The method according to claim 46, wherein at least exon 14 of the RB1 gene is quantitatively amplified, and wherein the primers used are GTGATTTTCT AAAATAGCAG GC [Seq ID No.: 41] and CCAGGATGAT CTTGATGCC [Seq ID No.: 42]. 67. The method according to claim 46, wherein at least exons 15 and 16 of the RB1 gene are quantitatively amplified using a single pair of primers, and wherein the primers used are CAATGCTGAC ACAAATAAGG TT [Seq ID No.: 49] and CCCCCGACCA AAGAAACACA [Seq ID No.: 50]. 68. The method according to claim 46, wherein at least exon 17 of the RB1 gene is quantitatively amplified, and wherein the primers used are ACCTTTCTAC TGTTTTCTTT GT [Seq ID No.: 51] and AAACACCTCT CACTAACAAT [Seq ID No.: 52]. 69. The method according to claim 46, wherein at least exon 18 of the RB1 gene is quantitatively amplified, and wherein the primers used are TTTTTGTGTG TGGGAAGTAC A [Seq ID No.: 17] and ATTCTATTCC CTACAGTTTC TT [Seq ID No.: 18]. 70. The method according to claim 46, wherein at least exon 19 of the RB1 gene is quantitatively amplified, and wherein the primers used are TGTATAATCT GTGATTCTTA GC [Seq ID No.: 53] and GCAACATTAT CATTTCCATT TT [Seq ID No.: 54]. 71. The method according to claim 46, wherein at least exon 20 of the RB1 gene is quantitatively amplified, and wherein the primers used are GAAAAGAGTG GTAGAAAAGA GG [Seq ID No.: 43] and TAACAAGTAA GTAGGGAGGA GA [Seq ID No.: 44]. 72. The method according to claim 46, wherein at least exon 21 of the RB1 gene is quantitatively amplified, and wherein the primers used are GGCTAAAAGA AAGAAAATGG [Seq ID No.: 19] and TTACCTATGT TATGTTATGG [Seq ID No.: 20]. 73. The method according to claim 46, wherein at least exon 22 of the RB1 gene is quantitatively amplified, and wherein the primers used are TATGTGCTTC TTACCAGTCA AA [Seq ID No.: 21] and GGAGTCATTT TTGTTGGTGT TG [Seq ID No.: 22]. 74. The method according to claim 46, wherein at least exon 23 of the RB1 gene is quantitatively amplified, and wherein the primers used are AATCTAATGT AATGGGTCCA CC [Seq ID No.: 23] and ATCAAAATAA TCCCCCTCTC AT [Seq ID No.: 24]. 75. The method according to claim 46, wherein at least exon 24 of the RB1 gene is quantitatively amplified, and wherein the primers used are GTATTTATGC TCATCTCTGC [Seq ID No.: 45] and GTGTTTGAAT AACTGCATTT GG [Seq ID No.: 46]. 76. The method according to claim 46, wherein at least exon 25 of the RB1 gene is quantitatively amplified, and wherein the primers used are TCAAACTATA ACTTGAGGTT GC [Seq ID No.: 9] and AAAGAAATTG GTATAAGCCA GG [Seq ID No.: 10]. 77. The method according to claim 46, wherein at least exon 26 of the RB1 gene is quantitatively amplified, and wherein the primers used are CGAAAGCATC ATAGTTACTG G [Seq ID No.: 47] and ATATAACGAA AAGACTTCTT GC [Seq ID No.: 48]. 78. The method according to claim 46, wherein at least exon 27 of the RB1 gene is quantitatively amplified, and wherein the primers used are ACTTACCCAG TACCATCAAT GC [Seq ID No.: 37] and TCAAGTGGCT TAGGAATCAC CC [Seq ID No.: 38]. 79. The method according to claim 46, wherein exons 2, 3, 5, 13 and 25 of the sample RB1 gene are coamplifed in a single reaction. 80. The method according to claim 79, wherein the primers employed for the coamplification are as follows: for exon 2, Seq.ID No.: 1 and Seq.ID No.: 2; for exon 3, Seq.ID No.: 3 and Seq.ID No.: 4; for exon 5, Seq.ID No.: 5 and Seq.ID No.: 6; for exon 13, Seq.ID No.: 7 and Seq.ID No.: 8; and for exon 25, Seq.ID No.: 9 and Seq.ID No.: 10. 81. The method according to claim 46, wherein exons 1, 8, 18, 21, 22, and 23 of the sample RB1 gene are coamplified in a single reaction. 82. The method according to claim 81, wherein the primers employed for the coamplification are as follows: for exon 1, Seq.ID No.: 13 and Seq.ID No.: 14; for exon 8, Seq.ID No.: 15 and Seq.ID No.: 16; for exon 18, Seq.ID No.: 17 and Seq.ID No.: 18; for exon 21, Seq.ID No.: 19 and Seq.ID No.: 20; for exon 22, Seq.ID No.: 21 and Seq. I.D No.: 22; and for exon 23, Seq.ID No.: 23 and Seq.ID No.: 24. 83. The method according to claim 46, wherein exons 6, 7, 8, 9, 10, 11, 12, and 27 of the sample RB1 gene are coamplified in a single reaction. 84. The method according to claim 83, wherein the primers employed for the coamplification are as follows: for exon 6, Seq.ID No.: 25 and Seq.ID No.: 26; for exon 7, Seq.ID No.: 27 and Seq.ID No.: 28; for exon 9, Seq.ID No.: 29 and Seq.ID No.: 30; for exon 10, Seq.ID No.: 31 and Seq.ID No.: 32; for exon 11, Seq.ID. No.: 33 and Seq.ID No.: 34; for exon 12, Seq.ID No.: 35 and Seq.ID No.: 36; and for exon 27, Seq.ID No.: 3.7 and Seq.ID No.: 38. 85. The method according to claim 46, wherein exons 4, 14, 20, 24 and 26 of the sample RB1 gene are coamplified in a single reaction. 86. The method according to claim 85, wherein the primers employed for the coamplification are as follows: for exon 4, Seq.ID No.: 39 and Seq.ID No.: 40; for exon 14, Seq.ID No.: 41 and Seq.ID No.: 42; for exon 20, Seq.ID No.: 43 and Seq.ID No.: 44; for exon 24, Seq.ID No.: 45 and Seq.ID No.: 46; and for exon 26, Seq.ID No.: 47 and Seq.ID No.: 48. 87. The method according to claim 46, wherein exons 15, 16, 17, and 19 of the sample RB1 gene are coamplified in a single reaction. 88. The method according to claim 87, wherein the primers employed for the coamplification are as follows: for exons 15 and 16, Seq.ID No.: 49 and Seq.ID No.: 50; for exon 17, Seq.ID No.: 51 and Seq.ID No.: 52; and for exon 19, Seq.ID No.: 53 and Seq.ID No.: 54. 89. The method for generating a report on the nature of a mutation causing retinoblastoma in a patient, comprising the steps of (a) obtaining a sample of patient tissue, (b) quantitatively amplifying one or more exons of the sample retinoblastoma gene using primers complementary to intron regions immediately flanking each amplified exon; (c) determining the lengths of the amplification products for each amplified sample exon and comparing that length to the length of amplification products obtained when a wild-type retinoblastoma gene is amplified using the same primers, whereby differences in length between an amplified sample exon and the corresponding amplified wild-type exon reflect the occurrence on an insertion or deletion mutation in the sample retinoblastoma gene; (d) determining the nucleic acid sequence of each exon identified in step (c) to contain an insertion or deletion mutation, or in the event no insertion or deletion mutations are identified, the nucleic acid sequence of at least one exon until all exons have been sequenced or a mutation has been detected; and (e) generating a report identifying the exon in which the mutation is located. 90. A method according to claim 89, wherein the report is a printed report. 91. A method according to claim 89, wherein the report is an electronic communication. 92. A method according to claim 89, wherein the report is a data entry in a computer record relating to the patient. 93. A method for identifying mutations in a plurality of samples containing the retinoblastoma gene comprising the steps of: (a) quantitatively amplifying one or more exons of the retinoblastoma gene of each sample using primers complementary to intron regions immediately flanking each amplified exon; (b) determining the lengths of the amplification products for each amplified exon of each sample and comparing that length to the length of amplification products obtained when a wild-type retinoblastoma gene is amplified using the same primers, whereby differences in length between an amplified sample exon and the corresponding amplified wild-type exon reflect the occurrence on an insertion or deletion mutation in the sample retinoblastoma gene; and (c) for each sample for which no insertion or deletion mutations is identified, determining the complete the nucleic acid sequence of at least one exon of the retinoblastoma gene. 94. The method according to claim 93, further comprising the step of determining the quantity of the amplification products for each sample and comparing this quantity to that of the corresponding amplified wild-type exon. 95. The method according to claim 93, wherein at least one exon selected from consisting of exons 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24 is sequenced for each sample. 96. The method according to claim 93, wherein the lengths of the amplification products for each amplified exon of each sample are determined by electrophoresis on a gel having a resolution of one-base. 97. The method according to claim 93, wherein the lengths of the amplification products for each amplified exon of each sample are determined by electrophoresis on a polyacrylamide gel having a resolution of one-base. 98. The method according to claim 93, wherein at least two of the exons of the retinoblastoma gene of each sample are coamplified in a single reaction. 99. The method according to claim 93, wherein at least three exons of the retinoblastoma gene of each sample are coamplified in a single reaction. 100. The method according to claim 99, wherein the primers for the coamplified exons of the retinoblastoma gene are selected to provide amplified fragments of different lengths. 101. The method according to claim 100, wherein lengths of the amplified fragments differ by at least 5 bases. 102. The method according to claim 93, wherein at least exon 1 of the retinoblastoma gene is quantitatively amplified, and wherein the primers used are GCCCCAGTTC CCCACAGAC [Seq ID No.: 13] and ACCCCTCGCC CAAGAACCCA [Seq ID No. 14]. 103. The method according to claim 93, wherein at least exon 2 of the RB1 gene is quantitatively amplified, and wherein the primers used are ACTGTGTGGT ATCCTTATTT TG [Seq ID No.: 1] and ATAGTGATTT GAAGTTGGTT TTA [Seq ID No.: 2]. 104. The method according to claim 93, wherein at least exon 3 of the RB1 gene is quantitatively amplified, and wherein the primers used are ATACAGTTTT AACATAGTAT CCA [Seq ID No.: 3] and AAGTCTATTG AGAGGAAAAT CC [Seq ID No.: 4]. 105. The method according to claim 93, wherein at least exon 4 of the RB1 gene is quantitatively amplified, and wherein the primers used are TTGAAAACGA AATAACAC [Seq ID No.: 39] and ATAAAAAATC AGAGTGTAAC CC [Seq ID No.: 40]. 106. The method according to claim 93, wherein at least exon 5 of the RB1 gene is quantitatively amplified, and wherein the primers used are CTACTATGAC TTCTAAATTA CG [Seq ID No.: 5] and TCAAGATGTT TGAGATTATT CC [Seq ID No.: 6]. 107. The method according to claim 93, wherein at least exon 6 of the RB1 gene is quantitatively amplified, and wherein the primers used are AAAGAAACAC CCAAAAGATA [Seq ID No.: 25] and TAATAAGCCA AGCAGAGAAT GA [Seq ID No.: 26]. 108. The method according to claim 93, wherein at least exon 7 of the RB1 gene is quantitatively amplified, and wherein the primers used are TTATGGATAT ACTCTACCCT GC [Seq ID No.: 27] and CCTCCATTTG TATGTTATGG AC [Seq ID No.: 28]. 109. The method according to claim 93, wherein at least exon 8 of the RB1 gene is quantitatively amplified, and wherein the primers used are TCTAATGAAA CCTAATAAGT A [Seq ID No.: 15] and TGCTCATAAC AAAAGAAGTA A [Seq ID No.: 16]. 110. The method according to claim 93, wherein at least exon 9 of the RB1 gene is quantitatively amplified, and wherein the primers used are TCAAGAGTCA AGAGATTAGA [Seq ID No.: 29] and ATTATCCTCC CTCCACAGTC TC [Seq ID No.: 30]. 111. The method according to claim 93, wherein at least exon 10 of the RB1 gene is quantitatively amplified, and wherein the primers used are GTGCTGAGAG ATGTAATGA [Seq ID No.: 31] and TATCTAAAGG TCACTAAGC [Seq ID No.: 32]. 112. The method according to claim 93, wherein at least exon 11 of the RB1 gene is quantitatively amplified, and wherein the primers used are TGAGACAACA GAAGCATTAT [Seq ID No.: 33] and TGAACAAATC TGAAACACTA T [Seq ID No.: 34]. 113. The method according to claim 93, wherein at least exon 12 of the RB1 gene is quantitatively amplified, and wherein the primers used are CTCCCTTCAT TGCTTAACAC AT [Seq ID No.: 35] and AAAAGCAAGA AAAGATTATG G [Seq ID No.: 36]. 114. The method according to claim 93, wherein at least exon 13 of the RB1 gene is quantitatively amplified, and wherein the primers used are TGCTTATGTT CAGTAGTTGT G [Seq ID No.: 7] and TAATGGGGTG GGAGGTAGTT T [Seq ID No.: 8]. 115. The method according to claim 93, wherein at least exon 14 of the RB1 gene is quantitatively amplified, and wherein the primers used are GTGATTTTCT AAAATAGCAG GC [Seq ID No.: 41] and CCAGGATGAT CTTGATGCC [Seq ID No.: 42]. 116. The method according to claim 93, wherein at least exons 15 and 16 of the RB1 gene are quantitatively amplified using a single pair of primers, and wherein the primers used are CAATGCTGAC ACAAATAAGG TT [Seq ID No.: 49] and CCCCCGACCA AAGAAACACA [Seq ID No.: 50]. 117. The method according to claim 93, wherein at least exon 17 of the RB1 gene is quantitatively amplified, and wherein the primers used are ACCTTTCTAC TGTTTTCTTT GT [Seq ID No.: 51 ] and AAACACCTCT CACTAACAAT [Seq ID No.: 52]. 118. The method according to claim 93, wherein at least exon 18 of the RB1 gene is quantitatively amplified, and wherein the primers used are TTTTTGTGTG TGGGAAGTAC A [Seq ID No.: 17] and ATTCTATTCC CTACAGTTTC TT [Seq ID No.: 18]. 119. The method according to claim 93, wherein at least exon 19 of the RB1 gene is quantitatively amplified, and wherein the primers used are TGTATAATCT GTGATTCTTA GC [Seq ID No.: 53] and GCAACATTAT CATTTCCATT TT [Seq ID No.: 54]. 120. The method according to claim 93, wherein at least exon 20 of the RB1 gene is quantitatively amplified, and wherein the primers used are GAAAAGAGTG GTAGAAAAGA GG [Seq ID No.: 43] and TAACAAGTAA GTAGGGAGGA GA [Seq ID No.: 44]. 121. The method according to claim 93, wherein at least exon 21 of the RB1 gene is quantitatively amplified, and wherein the primers used are GGCTAAAAGA AAGAAAATGG [Seq ID No.: 19] and TTACCTATGT TATGTTATGG [Seq ID No.: 20]. 122. The method according to claim 93, wherein at least exon 22 of the RB1 gene is quantitatively amplified, and wherein the primers used are TATGTGCTTC TTACCAGTCA AA [Seq ID No.: 21] and GGAGTCATTT TTGTTGGTGT TG [Seq ID No.: 22]. 123. The method according to claim 93, wherein at least exon 23 of the RB1 gene is quantitatively amplified, and wherein the primers used are AATCTAATGT AATGGGTCCA CC [Seq ID No.: 23] and ATCAAAATAA TCCCCCTCTC AT [Seq ID No.: 24]. 124. The method according to claim 93, wherein at least exon 24 of the RB1 gene is quantitatively amplified, and wherein the primers used are GTATTTATGC TCATCTCTGC [Seq ID No.: 45] and GTGTTTGAAT AACTGCATTr GG [Seq ID No.: 46]. 125. The method according to claim 93, wherein at least exon 25 of the RB1 gene is quantitatively amplified, and wherein the primers used are TCAAACTATA ACTTGAGGTT GC [Seq ID No.: 9] and AAAGAAATTG GTATAAGCCA GG [Seq ID No.: 10]. 126. The method according to claim 93, wherein at least exon 26 of the RB1 gene is quantitatively amplified, and wherein the primers used are CGAAAGCATC ATAGTTACTG G [Seq ID No.: 47] and ATATAACGAA AAGACTTCTT GC [Seq ID No.: 48]. 127. The method according to claim 93, wherein at least exon 27 of the RB1 gene is quantitatively amplified, and wherein the primers used are ACTTACCCAG TACCATCAAT GC [Seq ID No.: 37] and TCAAGTGGCT TAGGAATCAC CC [Seq ID No.: 38]. 128. The method according to claim 93, wherein exons 2, 3, 5, 13 and 25 of the sample RB1 gene are coamplified in a single reaction. 129. The method according to claim 128, wherein the primers employed for the coamplification are as follows: for exon 2, Seq.ID No.: 1 and Seq.ID No.: 2; for exon 3, Seq.ID No.: 3 and Seq.ID No.: 4; for exon 5, Seq.ID No.: 5 and Seq.ID No.: 6; for exon 13, Seq.ID No.: 7 and Seq.ID No.: 8; and for exon 25, Seq.ID No.: 9 and Seq.ID No.: 10. 130. The method according to claim 93, wherein exons 1, 8, 18, 21, 22, and 23 of the sample RB1 gene are coamplified in a single reaction. 131. The method according to claim 168, wherein the primers employed for the coamplification are as follows: for exon 1, Seq.ID No.: 13 and Seq.ID No.: 14; for exon 8, Seq.ID No.: 15 and Seq.ID No.: 16; for exon 18, Seq.ID No.: 17 and Seq.ID No.: 18; for exon 21, Seq.ID No.: 19 and Seq.ID No.: 20; for exon 22, Seq.ID No.: 21 and Seq.ID No.: 22; and for exon 23, Seq.ID No.: 23 and Seq.ID No.: 24. 132. The method according to claim 93, wherein exons 6, 7, 8, 9, 10, 11, 12, and 27 of the sample RB1 gene are coamplified in a single reaction. 133. The method according to claim 132, wherein the primers employed for the coamplification are as follows: for exon 6, Seq.ID No.: 25 and Seq.ID No.: 26; for exon 7, Seq.ID No.: 27 and Seq.ID No.: 28; for exon 9, Seq.ID No.: 29 and Seq.ID No.: 30; for exon 10, Seq.ID No.: 31 and Seq.ID No.: 32; for exon 11, Seq.ID No.: 33 and Seq.ID No.: 34; for exon 12, Seq.ID No.: 35 and Seq.ID No.: 36; and for exon 27, Seq.ID No.: 37 and Seq.ID No.: 38. 134. The method according to claim 93, wherein exons 4, 14, 20, 24 and 26 of the sample RB1 gene are coamplified in a single reaction. 135. The method according to claim 134, wherein the primers employed for the coamplification are as follows: for exon 4, Seq.ID No.: 39 and Seq.ID No.: 40; for exon 14, Seq.ID No.: 41 and Seq.ID No.: 42; for exon 20, Seq.ID No.: 43 and Seq.ID No.: 44; for exon 24, Seq.ID No.: 45 and Seq.ID No.: 46; and for exon 26, Seq.ID No.: 47 and Seq.ID No.: 48. 136. The method according to claim 93, wherein exons 15, 16, 17, and 19 of the sample RB1 gene are coamplified in a single reaction. 137. The method according to claim 136, wherein the primers employed for the coamplification are as follows: for exons 15 and 16, Seq.ID No.: 49 and Seq.ID No.: 50; for exon 17, Seq.ID No.: 51 and Seq.ID No.: 52; and for exon 19, Seq.ID No.: 53 and Seq.ID No.: 54.

Details for Patent 5,550,020

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2039-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2039-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2039-02-26