Nucleic acid amplification primers for PCR-based clonality studies
The invention relates to PCR-based clonality studies for among others early diagnosis of lymphoproliferative disorders. Provided is a set of nucleic acid amplification primers comprising a forward primer, or a variant thereof, and a reverse primer, or a variant thereof, capable of amplifying a rearrangement selected from the group consisting of a VH-JH IGH rearrangement, a DH-JH IGH rearrangement, a VK-JK IGK rearrangement, a VK/intron-Kde IGK rearrangement, a Vλ-Jλ IGL rearrangement, a Vβ-Jβ TCRB rearrangement, a Dβ-Jβ TCRB rearrangement, a Vγ-Jγ TCRG rearrangement, a Vδ-Jδ TCRD rearrangement, a Dδ-Dδ TCRD rearrangement, a Dδ-Jδ TCRD rearrangement, a Vδ-Dδ TCRD rearrangement, or a translocation selected from t(11;14)(BCL1-IGH) and t(14;18)(BCL2-IGH). The primers can be used in PCR-based clonality studies for early diagnosis of lymphoproliferative disorders and detection of minimal residual disease (MRD). Also provided is a kit comprising at least one set of primers of the invention.
1. A kit for the detection of at least one IGH rearrangement, wherein said kit comprises a plurality of forward primers selected from the group consisting of SEQ ID NOs: 1-8, 10-15, 17-20, and 22-28, and at least one reverse primer comprising SEQ ID NO:21, wherein at least one of said plurality of forward primers or at least one of said at least one reverse primer comprises a fluorescent label.
2. The kit of claim 1 further comprising forward primers comprising SEQ ID NOs: 9 and 16.
3. The kit of claim 1 , further comprising a set of nucleic amplification primers capable of amplifying a V K -J K IGK rearrangement comprising one or more forward primers of a sequence selected from SEQ ID NOs: 29-34 and one or more reverse primers of a sequence selected from SEQ ID NOs: 35 and 36.
4. The kit of claim 1 , further comprising a set of nucleic amplification primers capable of amplifying a V K /intron-Kde IGK rearrangement comprising one or more forward primers of a sequence selected from SEQ ID NOs: 29-34, 38 and a reverse primer of SEQ ID NO: 37.
5. The kit of claim 1 , further comprising a set of nucleic amplification primers capable of amplifying a Vλ-Jλ IGL rearrangement comprising one or more forward primers of a sequence selected from SEQ ID NOs: 39 and 40 and a reverse primer of SEQ ID NO: 41.
6. The kit of claim 1 , further comprising a set of nucleic amplification primers capable of amplifying a Vβ-Jβ TCRB rearrangement comprising one or more forward primers of a sequence selected from SEQ ID NOs: 42-64 and one or more reverse primers of a sequence selected from SEQ ID NOs: 65-77.
7. The kit of claim 1 , further comprising a set of nucleic amplification primers capable of amplifying a Dβ-Jβ TCRB rearrangement comprising one or more forward primers of a sequence selected from SEQ ID NOs: 78 and 79 and one or more reverse primers of a sequence selected from SEQ ID NOs: 65-77.
8. The kit of claim 1 , further comprising a set of nucleic amplification primers capable of amplifying a V γ -J γ TCRG rearrangement comprising one or more forward primers of a sequence selected from SEQ ID NOs: 80-83 and one or more reverse primers of a sequence selected from SEQ ID NOs: 84 and 85.
9. The kit of claim 1 , further comprising a set of nucleic amplification primers capable of amplifying a Vδ-Jδ TCRD rearrangement comprising one or more forward primers of a sequence selected from SEQ ID NOs: 86-91 and one or more reverse primers of a sequence selected from SEQ ID NOs: 92-95.
10. The kit of claim 1 , further comprising a set of nucleic amplification primers capable of amplifying a Dδ-Dδ TCRD rearrangement comprising a forward primer of SEQ ID NO: 96 and a reverse primer of SEQ ID NO: 97.
11. The kit of claim 1 , further comprising a set of nucleic amplification primers capable of amplifying a Dδ-Jδ TCRD rearrangement comprising a forward primer of SEQ ID NO: 96 and one or more reverse primers of a sequence selected from SEQ ID NOs: 92-95.
12. The kit of claim 1 , further comprising a set of nucleic amplification primers capable of amplifying a Vδ-Dδ TCRD rearrangement comprising one or more forward primers of a sequence selected from SEQ ID NOs: 86-91 and a reverse primer of SEQ ID NO: 97.
13. The kit of claim 1 , further comprising a set of nucleic amplification primers capable of amplifying a chromosomal translocation (11;14) (BCL1-IGH) comprising a forward primer of SEQ ID NO: 98 and a reverse primer of SEQ ID NO: 21.
14. The kit of claim 1 , further comprising a set of nucleic amplification primers capable of amplifying a chromosomal translocation t(14;18) (BCL2-IGH), comprising one or more forward primers of a sequence selected from SEQ ID NO: 100-109 and a reverse primer of SEQ ID NO: 21.
15. The kit of claim 1 , further comprising one or more primers sets of SEQ ID NOs: 111 and 112, SEQ ID NOs: 113 and 114, SEQ ID NOs: 115 and 116, SEQ ID NOs: 117 and 118, and SEQ ID NOs: 119 and 120.
16. The kit of claim 1 , further comprising forward primers comprising SEQ ID NOs: 29-34, 38-40, 42-64, 78-83, 86-91, 96, 98, 100-109 and reverse primers comprising SEQ ID NOs: 35-37, 41, 65-77, 84, 85, 92-95, and 97.
17. A method for assaying for two or more rearrangements, two or more translocations or at least one rearrangement and at least one translocation selected from the group consisting of a V H -J H IGH rearrangement, a D H -J H IGH rearrangement, a V K -J K IGK rearrangement, a V K /intron-Kde IGK rearrangement, a Vλ-Jλ IGL rearrangement, a Vβ-Jβ TCRB rearrangement, a Dβ-Jβ TCRB rearrangement, a V Y -J Y TCRG rearrangement, a Vδ-Jδ TCRD rearrangement, a Dδ-Dδ TCRD rearrangement, a Dδ-Jδ TCRD rearrangement, a Vδ-Dδ TCRD rearrangement, a t(11;14) (BCL1-IGH) translocation and t(14;18) (BCL2-IGH) translocation, using primers from the kit of claim 16 to amplify the corresponding locus in a sample.
18. A method according to claim 17 for the detection of minimal residual disease (MRD) or for identification of PCR targets to be used for MRD detection.
19. A method according to claim 18 , wherein an amplified nucleic acid is detected using automated high resolution PCR fragment analysis.
20. The kit according to claim 16 , further comprising primers comprising SEQ ID NOs: 111-120.
21. A method for detecting a D H -J H IGH rearrangement, comprising using the kit according to claim 1 to amplify the IGH locus in a sample.
22. The method of claim 21 , wherein said IGH locus is amplified in a PCR assay.
23. The method of claim 22 , wherein said PCR assay is a multiplex PCR assay.
24. A method for the detection of minimal residual disease (MRD) or for identification of PCR targets to be used for MRD detection comprising utilizing the primers of the kit of claim 1 to amplify nucleic acid in a sample.
25. A method for detecting a V H -J H IGH rearrangement, comprising using a primer kit comprising a plurality of forward primers selected from the group consisting of SEQ ID NOs: 1-8, 10-15, 17-20, and 22-28, and at least one reverse primer comprising SEQ ID NO:21 to amplify the IGH locus in a sample; wherein at least one of said plurality of forward primers or at least one of said at least one reverse primer comprises a fluorescent label.