Method and reagents for testing for mutations in the BRCA1 gene

• US 6,403,303 B1
• Filed: 05/14/1996
• Issued: 06/11/2002
• Est. Priority Date: 05/14/1996
• Status: Expired due to Term

• Current Assignee: Siemens Healthcare Diagnostics Incorporated (Siemens AG)
• Original Assignee: Visible Genetics, Inc. (Bayer AG)
• Inventors: Dunn, James M., Shipman, Robert, Leushner, James
• Primary Examiner(s): ARTHUR, LISA BENNETT
• Application Number:
  US08/649,950
• Time in Patent Office: 2,219 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 536/24.3, 536/24.33
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

Samples are tested for mutations in the BRCA1 gene using a hierarchical approach. First, each sample is amplified in one or more multiplex PCR amplification reactions. Each multiplex PCR reaction produces a mixture of amplified fragments. The sizes and amounts of these fragments are evaluated and compared to standard values reflecting the sizes and amounts of fragments produced when the same multiplex amplification is performed on the wild-type BRCA1 gene. Differences between the observed fragment sizes and/or amounts and those for the wild-type gene are indicative of a mutation with the BRCA1 gene of the sample. Next, one or more of the exons of the BRCA1 gene are sequenced, preferably only for those samples where no mutation was detected by analysis of the multiplex PCR fragments. The sequencing procedure can be performed by amplification and sequencing of the multiplex amplification mixture.

Claims

1. A method for testing a sample for mutations in the BRCA1 gene comprising the steps of:

(a) amplifying exons 1 and 21, or a portion of each exon, of the BRCA1 gene to produce amplified fragments, wherein the exons or portions thereof are amplified in a multiplex amplification reaction to produce multiplex amplification products for analysis;

(b) determining the sizes and amounts of amplified fragments in the multiplex amplification products and comparing the determined sizes or amounts to standard values for amplification of the same exons or portions thereof of wild-type BRCA1 gene, wherein a difference in fragment size or amount is indicative of the presence of a mutation in the BRCA1 gene; and

(c) if no mutation is detected in the BRCA1 gene as a result of the determination of the sizes and amounts of the amplification fragments, determining the sequence of one or more exons of the BRCA1 gene.

2. The method according to claim 1, wherein exons 1 and 21 or the portions thereof are amplified using the primers identified by Sequence ID Nos. 1, 2, 67 and 68.

3. A method for testing a sample for mutations in the BRCA1 gene comprising the steps of:

(a) amplifying exons 2, 5, 9 and 14, or a portion of each exon, of the BRCA1 gene to produce amplified fragments, wherein the exons or portions thereof are amplified using the primers identified by Sequence Nos. 3, 4, 9, 10, 17, 18, 53 and 54 in a multiplex amplification reaction to produce multiplex amplification products for analysis;

(b) determining the sizes and amounts of amplified fragments in the multiplex amplification products and comparing the determined sizes or amounts to standard values for amplification of the same exons or portions thereof of wild-type BRCA1 gene, wherein a difference in fragment size or amount is indicative of the presence of a mutation in the BRCA1 gene; and

(c) if no mutation is detected in the BRCA1 gene as a result of the determination of the sizes and amounts of the amplification fragments, determining the sequence of one or more exons of the BRCA1 gene.

4. A method for testing a sample for mutations in the BRCA1 gene comprising the steps of:

(a) amplifying exons 3, 7 and 15, or a portion of each exon, of the BRCA1 gene to produce amplified fragments, wherein the exons or portion thereof are amplified using the primers identified by Sequence Nos. 5, 6, 13, 14, 55 and 56 in a multiplex amplification reaction to produce multiplex amplification products for analysis;

(b) determining the sizes and amounts of amplified fragments in the multiplex amplification products and comparing the determined sizes or amounts to standard values for amplification of the same exons or portion thereof of wild-type BRCA1 gene, wherein a difference in fragment size or amount is indicative of the presence of a mutation in the BRCA1 gene; and

(c) if no mutation is detected in the BRCA1 gene as a result of the determination of the sizes and amounts of the amplification fragments, determining the sequence of one or more exons of the BRCA1 gene.

5. A method for testing a sample for mutations in the BRCA1 gene comprising the steps of:

(a) amplifying exons 6, 10, 17 and 18, or a portion of each exon, of the BRCA1 gene to produce amplified fragments, wherein the exons or portions thereof are amplified in a multiplex amplification reaction to produce multiplex amplification products for analysis;

(b) determining the sizes and amounts of amplified fragments in the multiplex amplification products and comparing the determined sizes or amounts to standard values for amplification of the same exons or portions thereof of wild-type BRCA1 gene, wherein a difference in fragment size or amount is indicative of the presence of a mutation in the BRCA1 gene; and

(c) if no mutation is detected in the BRCA1 gene as a result of the determination of the sizes and amounts of the amplification fragments, determining the sequence of one or more exons of the BRCA1 gene.

6. The method according to claim 5, wherein exons 6, 10, 17 and 18 or portions thereof are amplified using the primers identified by Sequence ID Nos. 11, 12, 19, 20, 59, 60, 61 and 62.

7. A method for testing a sample for mutations in the BRCA1 gene comprising the steps of:

(a) amplifying exons 4, 12 and 16, or a portion of each exon, of the BRCA1 gene to produce amplified fragments, wherein the exons or portions thereof are amplified in a multiplex amplification reaction to produce multiplex amplification products for analysis;

(b) determining the sizes and amounts of amplified fragments in the multiplex amplification products and comparing the determined sizes or amounts to standard values for amplification of the same exons or portions thereof of wild-type BRCA1 gene, wherein a difference in fragment size or amount is indicative of the presence of a mutation in the BRCA1 gene; and

(c) if no mutation is detected in the BRCA1 gene as a result of the determination of the sizes and amounts of the amplification fragments, determining the sequence of one or more exons of the BRCA1 gene.

8. The method according to claim 7, wherein exons 4, 12 and 16 or portions thereof are amlplified using the primers identified by Sequence ID Nos. 7, 8, 49, 50, 57 and 58.

9. A method for testing a sample for mutations in the BRCA1 gene comprising the steps of:

(a) amplifying exons 8, 13, 19 and 24, or a portion of each exon, of the BRCA1 gene to produce amplified fragments, wherein the exons or portions thereof are amplified in a multiplex amplification reaction to produce multiplex amplification products for analysis;

(b) determining the sizes and amounts of amplified fragments in the multiplex amplification products and comparing the determined sizes or amounts to standard values for amplification of the same exons or portions thereof of wild-type BRCA1 gene, wherein a difference in fragment size or amount is indicative of the presence of a mutation in the BRCA1 gene; and

(c) if no mutation is detected in the BRCA1 gene as a result of the determination of the sizes and amounts of the amplification fragments, determining the sequence of one or more exons of the BRCA1 gene.

10. The method according to claim 9, wherein exons 8, 13, 19 and 24 or portions thereof are amplified using the primers identified by Sequence ID Nos. 15, 16, 51, 52, 63, 64, 73 and 74.

11. A method for testing a sample for mutations in the BRCA1 gene comprising the steps of:

(a) amplifying exons 20, 22 and 23, or portion of each exon, of the BRCA1 gene to produce amplified fragments, wherein the exons or portions thereof are amplified in a multiplex amplification reaction to produce multiplex amplification products for analysis;

(b) determining the sizes and amounts of amplified fragments in the multiplex amplification products and comparing the determined sizes or amounts to standard values for amplification of the same exons or portions thereof of wild-type BRCA1 gene, wherein a difference in fragment size or amount is indicative of the presence of a mutation in the BRCA1 gene; and

(c) if no mutation is detected in the BRCA1 gene as a result of the determination of the sizes and amounts of the amplification fragments, determining the sequence of one or more exons of the BRCA1 gene.

12. The method according to claim 11, wherein exons 20, 22 and 23 or portions thereof are amplified using the primers identified by Sequence ID Nos. 65, 66, 69, 70, 71 and 72.

13. A method for testing a sample for mutations in the BRCA1 gene comprising the steps of:

(a) amplifying exons 2 and 20, or a portion of each exon, of the BRCA1 gene to produce amplified fragments, wherein the exons or portions thereof are amplified using the primers identified by Sequence Nos. 3, 4, 65 and 66 in a multiplex amplification reaction to produce multiplex amplification products for analysis;

(b) determining the sizes and amounts of amplified fragments in the multiplex amplification products and comparing the determined sizes or amounts to standard values for amplification of the same exons or portions thereof of wild-type BRCA1 gene, wherein a difference in fragment size or amount is indicative of the presence of a mutation in the BRCA1 gene; and

(c) if no mutation is detected in the BRCA1 gene as a result of the determination of the sizes and amounts of the amplification fragments, determining the sequence of one or more exons of the BRCA1 gene.

14. A kit for testing a sample for mutations in the BRCA1 gene comprising a mixture of at least four oligonucleotide primers, said primers being selected to amplify exons 1 and 21, or a portion of each exon, of the BRCA1 gene in a multiplex amplification reaction.

15. The kit according to claim 14, wherein the primers for amplification of exons 1 and 21 are the primers identified by Sequence ID Nos. 1, 2, 69 and 70.

16. A kit for testing a sample for mutations in the BRCA1 gene comprising a mixture of at least four oligonucleotide primers, said primers being selected to amplify exons 2, 5, 9 and 14, or a portion of each exon, of the BRCA1 gene in a multiplex amplification reaction, wherein the primers used for amplification are the primers identified by Sequence ID Nos. 3, 4, 9, 10, 17, 18, 55 and 56.

17. A kit for testing a sample for mutations in the BRCA1 gene comprising a mixture of at least four oligonucleotide primers, said primers being selected to amplify exons 3, 7 and 15, or a portion of each exon, of the BRCA1 gene in a multiplex amplification reaction, wherein the primers used for amplification are the primers identified by Sequence ID Nos. 5, 6, 13, 14, 57 and 58.

18. A kit for testing a sample for mutations in the BRCA1 gene comprising a mixture of at least four oligonucleotide primers, said primers being selected to amplify exons 6, 10, 17 and 18, or a portion of each exon, of the BRCA1 gene in a multiplex amplification reaction.

19. The kit according to claim 18, wherein the primers for amplification of exons 6, 10, 17 and 18 are the primers identified by Sequence ID Nos. 11, 12, 19, 20, 61, 62, 63 and 64.

20. A kit for testing a sample for mutations in the BRCA1 gene comprising a mixture of at least four oligonucleotide primers, said primers being selected to amplify exons 4, 12 and 16, or a portion of each exon, of the BRCA1 gene in a multiplex amplification reaction.

21. The kit according to claim 20, wherein the primers for amplification of exons 4, 12 and 16 are the primers identified by Sequence ID Nos. 7, 8, 51, 52, 59 and 60.

22. A kit for testing a sample for mutations in the BRCA1 gene comprising a mixture of at least four oligonueleotide primers, said primers being selected to amplify exons 8, 13, 19 and 24, or a portion of each exon, of the BRCA1 gene in a multiplex amplification reaction.

23. The kit according to claim 22, wherein the primers for amplification of exons 8, 13, 19 and 24 are the primers identified by Sequence ID Nos. 15, 16, 53, 54, 65, 66, 75 and 76.

24. A kit for testing a sample for mutations in the BRCA1 gene comprising a mixture of at least four oligonucleotide primers, said primers being selected to amplify exons 20, 22 and 23, or a portion of each exon, of the BRCA1 gene in a multiplex amplification reaction.

25. The kit according to claim 24, wherein the primers for amplification of exons 20, 22 and 23 are the primers identified by Sequence ID Nos. 67, 68, 71, 72, 73 and 74.

26. A kit for testing a sample for mutations in the BRCA1 gene comprising a mixture of at least four oligonucleotide primers, said primers being selected to amplify exons 2 and 20, or a portion of each exon, of the BRCA1 gene in a multiplex amplification reaction, wherein the primers used for amplification are the primers identified by Sequence ID Nos. 3, 4, 65 and 66.

27. An oligonucleotide primer having the sequence as set forth in any one of Sequence ID Nos. 1 through 77.