Claims for Patent: 6,103,465

Summary for Patent: 6,103,465

Title:	Methods and reagents for typing HLA class I genes
Abstract:	A method for typing HLA class 1 genes. The method entails first contacting a sample DNA with first and second amplification primers, wherein the first and second primers are each at least partially located in an exonic region. Next, using the first and second primers, a target sequence is amplified by the PCR to form an amplicon of the target sequence. Finally, the amplicon is detected with a sequence-specific detection means, e.g., DNA sequencing. The invention also includes specific amplification primers, specific sequencing primers, and kits especially adapted for use with the above HLA typing method.
Inventor(s):	Johnston-Dow; Leslie (Palo Alto, CA), Chadwick; Robert B. (Dublin, OH), Parham; Peter (Stanford, CA)
Assignee:	The Perkin-Elmer Corporation (Foster City, CA)
Application Number:	08/538,666
Patent Claims:	1. A method useful for typing HLA class I genes comprising the steps of: providing a sample DNA containing a HLA class I gene having a first exon, a second exon, and a target sequence; wherein the HLA class I gene is selected from the group consisting of HLA-A gene, HLA-B gene, and HLA-C gene; wherein the first exon is exon 1 of the HLA class I gene and the second exon is exon 5 of the HLA class I gene; contacting the sample DNA with a first amplification primer, the first amplification primer including a sequence complementary to the first exon of the HLA class I gene; contacting the sample DNA with a second amplification primer, the second amplification primer including sequence complementary to the second exon of the HLA class I gene; amplifying the target sequence of the sample DNA by PCR using the first and second amplification primers, thereby forming an amplicon; and detecting the amplicon using a sequence-specific detection means. 2. The method of claim 1 wherein the HLA class I gene is HLA-A and the first amplification primer comprises the oligonucleotide sequence CGCCGAGGATGGCCGTC (SEQ ID #1) and the second amplification primer comprises the degenerate oligonucleotide sequences GGAGAACCAGGCCAGCAATGATGCCC (SEQ ID #2) and GGAGAACTAGGCCAGCAATGATGCCC (SEQ ID #3). 3. The method of claim 1 wherein the HLA class I gene is HLA-B and the first amplification primer comprises the degenerate oligonucleotide sequences CCTCCTGCTGCTCTCGGC (SEQ ID #21) and CCTCCTGCTGCTCTCGGG (SEQ ID#22), and GCTGCTCTGGGGGGCAG (SEQ ID #23), and the second amplification primer comprises the oligonucleotide sequence GCTCCGATGACCACAACTGCT (SEQ ID #24). 4. The method of claim 1 wherein the HLA class I gene is HLA-B and the first amplification primer comprises the oligonucleotide sequence GGCCCTGACCGAGACCTGG (SEQ ID #4) and the second amplification primer comprises the oligonucleotide sequence TCCGATGACCACAACTGCTAGGAC (SEQ ID #5). 5. The method of claim 1 wherein the HLA class I gene is HLA-C and the first amplification primer comprises the degenerate oligonucleotide sequences GGCCCTGACCGAGACCTGGGC (SEQ ID #6) and GGCCCTGACCCAGACCTGGGC (SEQ ID #7) and the second amplification primer comprises the oligonucleotide sequence CCACAGCTCCTAGGACAGCTAGGA (SEQ ID #8). 6. The method of claim 1 wherein the HLA class I gene is HLA-C and the first amplification primer comprises the oligonucleotide sequence CATCCTGCTGCTCTCGGGAG (SEQ ID #30) and the second amplification primer comprises the oligonucleotide sequence CCACAGCTCCTAGGACAGCTAGGA (SEQ ID #8). 7. The method of claim 1 wherein the sequence-specific detection means is DNA sequencing. 8. The method of claim 1 wherein the sequence-specific detection means is Sanger-type DNA sequencing comprising the steps of: contacting the amplicon with a sequencing primer; extending the sequencing primer using a polymerase in the presense of deoxynucleotides and dideoxynucleotides, thereby forming a mixture of extension products differing in length; separating the extension products such that extension products differing in length by a single nucleotide are resolved; and, detecting the separated extension products. 9. The method of claim 8 wherein the sequencing primer (i) is between 10 nucleotides and 30 nucleotides in length and (ii) has a nucleotide sequence complementary to the HLA-A, HLA-B, and HLA-C genes in the nucleotide sequence regions selected from the group consisting of: a first nucleotide sequence region from -20 nucleotides to +20 nucleotides of the 5' intron-exon border of the sense strand of exon 2; a second nucleotide sequence region from -30 nucleotides to +20 nucleotides of the 5' intron-exon border of the sense strand of exon 3; a third nucleotide sequence region from 31 30 nucleotides to +20 nucleotides of the 5' intron-exon border of the sense strand exon 4; a fourth nucleotide sequence region from +30 nucleotides to -20 nucleotides of the 5' intron-exon border of the antisense strand exon 2; a fifth nucleotide sequence region from +20 nucleotides to -20 nucleotides of the 5' intron-exon border of the antisense strand of exon 3; and a sixth nucleotide sequence region from +40 nucleotides to -10 nucleotides of the 5' intron-exon border of the antisense strand of exon 4. 10. The method of claim 9 wherein each member of the set of sequencing primers is selected from the group consisting of: a first sequencing primer having the sequence CTCGCCCCCAGGCTCCCAC (SEQ ID #15); a second sequencing primer having the sequence GCGGGGGCGGGTCCAGG (SEQ ID #16); a third sequencing primer having the sequence CTGACTCTTCCCATCAGACCC (SEQ ID #17); a fourth sequencing primer having the sequence CACTCACCGGCCTCGCTCTGG (SEQ ID #18); a fifth sequencing primer having the sequence CCACTGCCCCTGGTACCCG (SEQ ID #19); and a sixth sequencing primer having the sequence AGGGTGAGGGGCTTCGGCAGCC (SEQ ID #20). 11. The method of claim 8 wherein the HLA class 1 gene is the sense strand of exon 2 of the HLA-A gene and the sequencing primer comprises the degenerate oligonucleotide sequences TCGGGCAGGTCTCAGCC (SEQ ID#25) and TCGGGCGGGTCTCAGCC (SEQ ID #26). 12. The method of claim 8 wherein the HLA class 1 gene is the antisense strand of exon 2 of the HLA-A gene and the sequencing primer comprises the oligonucleotide sequence CACTCACCGGCCTCGCTCTGG (SEQ ID #12). 13. The method of claim 8 wherein the HLA class 1 gene is the sense strand of exon 3 of the HLA-A gene and the sequencing primer comprises the degenerate oligonucleotide sequences GGGCTCGGGGGACCGGG (SEQ ID #27) and GGGCTCGGGGGACTGGG (SEQ ID #28). 14. The method of claim 8 wherein the HLA class 1 gene is the sense strand of exon 2 of the HLA-B gene and the sequencing primer comprises the oligonucleotide sequence CTCGCCCCCAGGCTCCCAC (SEQ ID #9). 15. The method of claim 8 wherein the HLA class 1 gene is the antisense strand of exon 2 of the HLA-B gene and the sequencing primer comprises the oligonucleotide sequence CACTCACCGGCCTCGCTCTGG (SEQ ID #12). 16. The method of claim 8 wherein the HLA class 1 gene is the sense strand of exon 3 of the HLA-B gene and the sequencing primer comprises the oligonucleotide sequence GCGGGGGCGGGTCCAGG (SEQ ID #10). 17. The method of claim 8 wherein the HLA class 1 gene is the antisense strand of exon 3 of the HLA-B gene and the sequencing primer comprises the oligonucleotide sequence CCACTGCCCCTGGTACCCG (SEQ ID #13). 18. The method of claim 8 wherein the HLA class 1 gene is the sense strand of exon 2 of the HLA-C gene and the sequencing primer comprises the oligonucleotide sequence AGGAGGGTCGGGCGGGTCTCAG (SEQ ID #31). 19. The method of claim 8 wherein the HLA class 1 gene is the antisense strand of exon 2 of the HLA-C gene and the sequencing primer comprises the oligonucleotide sequence CACTCACCGGCCTCGCTCTGG (SEQ ID #12). 20. The method of claim 8 wherein the HLA class 1 gene is the antisense strand of exon 3 of the HLA-C gene and the sequencing primer comprises the oligonucleotide sequence CCACTGCCCCTGGTACCCG (SEQ ID #13). 21. The method of claim 1 further comprising the step of comparing the the determined DNA sequence with the DNA sequences of known HLA types. 22. A kit for useful for typing HLA class I genes comprising: an amplification reagent comprising: a thermostable polymerase; each of the A, G, C, and T deoxynucleotides; a buffer; and first and second amplification primers, each amplification primer being between 10 nucleotides and 30 nucleotides in length, (i) the first primer having a nucleotide sequence complementary to sequence located in exon 1 of the HLA-A gene, and (ii) the second primer having a nucleotide sequence complementary to sequence located in exon 5 of the HLA-A gene. 23. A kit useful for typing HLA class I genes comprising: an amplification reagent comprising: a thermostable polymerase; each of the A, G, C, and T deoxynucleotides; a buffer; and first and second amplification primers, each amplification primer being between 10 nucleotides and 30 nucleotides in length, (i) the first primer having a nucleotide sequence complementary to sequence located in exon 1 of the HLA-B gene, and (ii) the second primer having a nucleotide sequence complementary to sequence located in exon 5 of the HLA-B gene. 24. A kit useful for typing HLA class I genes comprising: an amplification reagent comprising: a thermostable polymerase; each of the A, G, C, and T deoxynucleotides; a buffer; and first and second amplification primers, each amplification primer being between 10 nucleotides and 30 nucleotides in length, (i) the first primer having a nucleotide sequence complementary to sequence located in exon 1 of the HLA-C gene, and (ii) the second primer having a nucleotide sequence complementary to sequence located in exon 5 of the HLA-C gene. 25. The kit of claim 22, 23, or 24 further comprising: a sequencing reagent comprising: a polymerase; each of the A, G, C, and T deoxynucleotides; each of the A, G, C, and T dideoxydeoxynucleotides; a buffer, and a set of sequencing sequencing primers wherein each member of the set of sequencing primers (i) is between 10 nucleotides and 30 nucleotides in length and (ii) has a nucleotide sequence complementary to the HLA-A, HLA-B, and HLA-C genes in the nucleotide sequence regions selected from the group consisting of: a first nucleotide sequence region from -20 nucleotides to +20 nucleotides of the 5' intron-exon border of the sense strand of exon 2; a second nucleotide sequence region from -30 nucleotides to +20 nucleotides of the 5' intron-exon border of he sense strand of exon 3; a third nucleotide sequence region from -30 nucleotides to +20 nucleotides of the 5' intron-exon border of the sense strand exon 4; a fourth nucleotide sequence region from +30 nucleotides to -20 nucleotides of the 5' intron-exon border of the antisense strand exon 2; a fifth nucleotide sequence region from +20 nucleotides to -20 nucleotides of the 5' intron-exon border of the antisense strand of exon 3; and a sixth nucleotide sequence region from +40 nucleotides to -10 nucleotides of the 5' intron-exon border of the antisense strand of exon 4. 26. A set of PCR amplification primers useful for amplification of the HLA-A class 1 gene comprising: a first primer comprising the oligonucleotide sequence CGCCGAGGATGGCCGTC (SEQ ID #1); and a second primer comprising the degenerate oligonucleotide sequences GGAGAACCAGGCCAGCAATGATGCCC (SEQ ID #2) and GGAGAACTAGGCCAGCAATGATGCCC (SEQ ID #3). 27. A set of PCR amplification primers useful for amplification of the HLA-B class 1 gene comprising: a first primer comprising the oligonucleotide sequence GGCCCTGACCGAGACCTGG (SEQ ID #4); and a second primer comprising the oligonucleotide sequence TCCGATGACCACAACTGCTAGGAC (SEQ ID #5). 28. A set of PCR amplification primers useful for amplification of the HLA-B class 1 gene comprising: a first primer comprising the degenerate oligonucleotide sequences CCTCCTGCTGCTCTCGGC (SEQ ID #21), and CCTCCTGCTGCTCTCGGGA (SEQ ID #22), and GCTGCTCTGGGGGGCAG (SEQ ID #23); and a second primer comprising the oligonucleotide sequence GCTCCGATGACCACAACTGCT (SEQ ID #24). 29. A set of PCR amplification primers useful for amplification of the HLA-C class 1 gene comprising: a first primer comprising the degenerate oligonucleotide sequences GGCCCTGACCCAGACCTGGGC (SEQ ID #6), and GGCCCTGACCCAGACCTGGGC (SEQ ID #7); and a second primer comprising the oligonucleotide sequence CCACAGCTCCTAGGACAGCTAGGA (SEQ ID #8). 30. A set of PCR amplification primers useful for amplification of the HLA-C class 1 gene comprising: a first primer comprising the oligonucleotide sequence CATCCTGCTGCTCTCGGGAG (SEQ ID #30); and a second primer comprising the oligonucleotide sequence CCACAGCTCCTAGGACAGCTAGGA (SEQ ID #8). 31. A sequencing primer useful for sequencing exon 2 of HLA-A, -B, or -C class 1 genes comprising the oligonucleotide sequence CTCGCCCCCAGGCTCCCAC (SEQ ID #9). 32. A sequencing primer useful for sequencing exon 2 of HLA-A, -B, or -C class 1 genes comprising the oligonucleotide sequence CACTCACCGGCCTCGCTCTGG (SEQ ID #12). 33. A sequencing primer useful for sequencing exon 2 of HLA-A, -B, or -C class 1 genes comprising the degenerate oligonucleotide sequences TCGGGCAGGTCTCAGCC (SEQ ID #25) and TCGGGCGGGTCTCAGCC (SEQ ID #26). 34. A sequencing primer useful for sequencing exon 2 of HLA-A, -B, or -C class 1 genes comprising the oligonucleotide sequence AGGAGGGTCGGGCGGGTCTCAG (SEQ ID #31). 35. A sequencing primer useful for sequencing exon 3 of HLA-A, -B, or -C class 1 genes comprising the oligonucleotide sequence GCGGGGGCGGGTCCAGG (SEQ ID #10). 36. A sequencing primer useful for sequencing exon 3 of HLA-A, -B, or -C class 1 genes comprising the oligonucleotide sequence CCACTGCCCCTGGTACCCG (SEQ ID #13). 37. A sequencing primer useful for sequencing exon 3 of HLA-A, -B, or -C class 1 genes comprising the oligonucleotide sequence GAGGCGCCCCGTGGC (SEQ ID #29). 38. A sequencing primer useful for sequencing exon 3 of HLA-A, -B, or -C class 1 genes comprising the degenerate oligonucleotide sequences GGGCTCGGGGGACCGGG (SEQ ID #27) and GGGCTCGGGGGACTGGG (SEQ ID #28). 39. A sequencing primer useful for sequencing exon 4 of HLA-A, -B, or -C class 1 genes comprising the oligonucleotide sequence CTGACTCTTCCCATCAGACCC (SEQ ID #11). 40. A sequencing primer useful for sequencing exon 4 of HLA-A, -B, or -C class 1 genes comprising the oligonucleotide sequence AGGGTGAGGGGCTTCGGCAGCC (SEQ ID #14). 41. A sequencing primer useful for sequencing exon 3 of HLA-A, -B, or -C class 1 genes comprising the oligonucleotide sequence GGGCTGACCACGGGGGCGGGGCCCAG (SEQ ID #32).

Details for Patent 6,103,465

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		2015-02-14
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2015-02-14
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2015-02-14