Assay for bcr/abl gene rearrangement

20050037373

10/742616

December 18, 2003

The present invention provides a simple high-throughput assay for detecting bcr/abl translocations. The method includes qualitative PCR methods for identifying the particular amplified translocation (e1a2 or b2a3/b3a2) and real time PCR for quantifying an amount of bcr/abl transcript (e1a2, b2a3 and b3a2). Quantitative measurement of bcr/abl transcript in accordance with the methods of the invention is useful for monitoring response to therapy.

Tseng, Richard W. (Mission Viejo, CA, US); Samoszuk, Michael K. (Rancho Santa Margarita, CA, US)

1. An oligonucleotide comprising no more than about 40 nucleotides in length, said oligonucleotide comprising a sequence consisting essentially of any one of SEQ ID NOs: 1-3 or a complementary sequence thereof.

2. An oligonucleotide of claim 1 wherein said sequence is any one of SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 3.

3. An oligonucleotide comprising no more than about 40 nucleotides in length, said oligonucleotide comprising a sequence consisting essentially of any one of SEQ ID NOs: 4 and 5 or a complementary sequence thereof.

4. An oligonucleotide of claim 3 wherein said sequence is SEQ ID NO. 4 or SEQ ID NO. 5.

5. An oligonucleotide comprising no more than about 40 nucleotides in length, said oligonucleotide comprising a sequence consisting essentially of any one of SEQ ID NOs: 6 and 7 or a complementary sequence thereof.

6. An oligonucleotide of claim 5 wherein said sequence is SEQ ID NO. 6 or SEQ ID NO. 7.

7. An oligonucleotide comprising no more than about 40 nucleotides in length, said oligonucleotide comprising a sequence consisting essentially of any one of SEQ ID NOs: 8-11 or a complementary sequence thereof.

8. An oligonucleotide of claim 7 wherein said sequence is SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, or SEQ ID NO. 11.

9. The oligonucleotide of claim 7 wherein the oligonucleotide is conjugated to a detectable label.

10. The oligonucleotide of claim 9 wherein the detectable label is a molecular beacon pair.

11. The oligonucleotide of claim 10 wherein the molecular beacon pair is 2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC), 6-carboxyfluorescein (FAM) or tetrachloro-6-carboxyfluorescein (TET), each in combination with a quencher moiety.

12. The oligonucleotide of claim 10 wherein the quencher moiety is tetra-methylcarboxyrhodamine (TAMRA) or 4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL).

13. The oligonucleotide of claim 10 wherein the sequence is 5′-ACACGACAACCGGGCAGTGCC-3′ (SEQ ID NO. 8) and the molecular beacon pair is FAM and a quencher moiety.

14. The oligonucleotide of claim 10 wherein the sequence is 5′-TGCTGTGGACAGTCTGGAGTTTCACACA-3′ (SEQ ID NO. 9) and the molecular beacon pair is FAM and a quencher moiety.

15. The oligonucleotide of claim 10 wherein the sequence is 5-CCA GTA GCA TCT GAC TTT GAG CCT CAG GG-3′ (SEQ ID NO. 10) and the molecular beacon pair is FAM and a quencher moiety.

16. The oligonucleotide of claim 10 wherein the sequence is 5′-CAA GCT TCC CGT TCT CAG CC-3′ (SEQ ID NO. 11) and the molecular beacon pair is FAM and a quencher moiety.

17. A method of determining if a bcr/abl translocation is present in a biological sample, comprising: a) contacting RNA or cDNA from the biological sample with oligonucleotide primers SEQ ID NO. 1-3 and with bcr/abl e1/a2 transcript probe SEQ ID NO: 8 and bcr/abl b2a2/b3a2 transcript probe SEQ ID NO: 9, wherein said probes are labeled with a molecular beacon pair; b) conducting amplification by temperature cycling with a DNA polymerase with 5′ exonuclease activity, wherein binding of the probe to amplified nucleic acid results in degradation of the probe during DNA synthesis; and c) monitoring the accumulation of amplified nucleic acid in real time by detecting an increase in reporter dye fluorescence over time, wherein an increase in reporter dye fluorescence indicates the presence of a bcr/abl translocation in the biological sample.

18. The method of claim 17 wherein the molecular beacon pair is 2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC), 6-carboxyfluorescein (FAM) or tetrachloro-6-carboxyfluorescein (TET) in combination with and a quencher moiety.

19. The method of claim 17 further comprising determining a level of a housekeeping gene transcript present in the biological sample, comprising: d) contacting the RNA or cDNA with a primer pair for amplifying a housekeeping gene transcript and a probe for the amplified housekeeping gene labeled with a molecular beacon pair; e) conducting amplification by temperature cycling with a DNA polymerase with 5′ exonuclease activity, wherein binding of the probe to amplified nucleic acid results in degradation of the probe during DNA synthesis; and f) monitoring the accumulation of amplified nucleic acid in real time by detecting an increase in reporter dye fluorescence over time, wherein an increase in reporter dye fluorescence indicates the presence of the housekeeping gene transcript in the biological sample.

20. The method of claim 19 further comprising the step of correlating the housekeeping gene transcript signal with the amount of a cell line RNA or cDNA that generates the same signal, said correlating a result of extrapolating the housekeeping gene signal from the biological sample to a standard curve of PCR signals versus cell line RNA or cDNA obtained by amplifying using steps d) and e) but with the cell line RNA or cDNA, respectively.

21. The method of claim 20 wherein the cell line RNA is from a leukemic cell line.

22. The method of claim 19 wherein said housekeeping gene transcript is abl or GAPDH.

23. The method of claim 19 wherein said housekeeping gene transcript is abl and said primer pair is SEQ ID NO: 4 and 5 and said probe is SEQ ID NO: 10.

24. The method of claim 19 wherein said housekeeping gene transcript is GAPDH and said primer pair is SEQ ID NO: 6 and 7 and said probe is SEQ ID NO: 11.

25. The method of claim 19 wherein said primer pair for amplifying a housekeeping gene transcript and said probe for the amplified housekeeping gene labeled with a molecular beacon pair are combined with oligonucleotide primers SEQ ID NO. 1-3 and bcr/abl e1/a2 transcript probe SEQ ID NO: 8 and bcr/abl b2a2/b3a2 transcript probe SEQ ID NO: 9 with RNA or cDNA from the biological sample and amplified together, wherein each probe is labeled with a different molecular beacon pair.

26. A method of determining if a blood or bone marrow sample contains CML or AML cells characterized in having a bcr/abl translocation, comprising, a) contacting RNA or cDNA from a biological sample with: i) oligonucleotide primers SEQ ID NO. 1 and 3 and bcr/abl e1/a2 transcript probe SEQ ID NO: 8, or ii) oligonucleotide primers SEQ ID NO: 2 and 3 and bcr/abl b2a2/b3a2 transcript probe SEQ ID NO: 9, wherein said probes are labeled with a molecular beacon pair; b) conducting amplification by temperature cycling with a DNA polymerase with 5′ exonuclease activity, wherein binding of the probe to amplified nucleic acid results in degradation of the probe during DNA synthesis; and c) monitoring the accumulation of amplified nucleic acid in real time by detecting an increase in reporter dye fluorescence over time, wherein an increase in reporter dye fluorescence indicates the presence of the particular bcr/abl translocation in the biological sample.

27. The method of claim 25, wherein if amplification is observed with SEQ ID NO: 2 and 3 and bcr/abl b2a2/b3a2 transcript probe SEQ ID NO: 9, the amplified product is evaluated by gel electrophoresis to distinguish the size of the amplified product wherein a size of about 124 bp indicates b2a2 and a size of about 199 bp indicates b3a2.

28. A method of monitoring response to therapy in a leukemia cancer patient characterized in having leukemic cells with a bcr/abl translocation genetic signature, comprising, determining a level of bcr/abl translocation correlating with leukemic cell number in blood or marrow of the patient before and after therapy, wherein the level bcr/abl translocation correlating with leukemic cell number in blood or marrow is determined by: a) contacting RNA or cDNA from blood or marrow of the patient with i) oligonucleotide primers SEQ ID NO. 1-3 and with bcr/abl e1/a2 transcript probe SEQ ID NO: 8 and bcr/abl b2a2/b3a2 transcript probe SEQ ID NO: 9 wherein said probes are labeled with a molecular beacon pair; or ii) oligonucleotide primers SEQ ID NO. 1 and 3 and bcr/abl e1/a2 transcript probe SEQ ID NO: 8, or ii) oligonucleotide primers SEQ ID NO: 2 and 3 and bcr/abl b2a2/b3a2 transcript probe SEQ ID NO: 9, and b) conducting amplification by temperature cycling with a DNA polymerase with 5′ exonuclease activity, wherein binding of the probe to amplified nucleic acid results in degradation of the probe during DNA synthesis; and c) monitoring the accumulation of amplified nucleic acid in real time by detecting an increase in reporter dye fluorescence over time to determine a signal of bcr/abl transcript in the sample; d) contacting RNA or cDNA RNA or cDNA from blood or marrow of the patient with a primer pair for amplifying a housekeeping gene transcript and a probe for the amplified housekeeping gene labeled with a molecular beacon pair; e) amplifying step d) by temperature cycling with a DNA polymerase with 5′ exonuclease activity, wherein binding of the probe to amplified nucleic acid results in degradation of the probe during DNA synthesis; and f) monitoring the accumulation of amplified nucleic acid in step e) in real time by detecting an increase in reporter dye fluorescence over time to determine a signal of the housekeeping gene transcript in the sample; g) obtaining a standard curve of amplified signals from serial dilutions of RNA or cDNA of a cell line using steps d)-f) but with the cell line RNA or cDNA in place of the patient RNA or cDNA; and h) extrapolating from the standard curve an amount of cell line RNA representing the amount of housekeeping gene transcript amplified from the patient sample, and calculating a ratio of bcr/abl transcript to the amount of cell line RNA representing the amount of housekeeping gene transcript amplified from the patient sample, wherein said ratio represents a level of bcr/abl transcript correlating with leukemic cell number in blood or marrow.

29. The method of claim 28 wherein the molecular beacon pair is 2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC), 6-carboxyfluorescein (FAM) or tetrachloro-6-carboxyfluorescein (TET) in combination with and a quencher moiety.

30. The method of claim 28 wherein said housekeeping gene transcript is abl or GAPDH.

31. The method of claim 28 wherein said housekeeping gene transcript is abl and said primer pair is SEQ ID NO: 4 and 5 and said probe is SEQ ID NO: 10.

32. The method of claim 28 wherein said housekeeping gene transcript is GAPDH and said primer pair is SEQ ID NO: 6 and 7 and said probe is SEQ ID NO: 11.

33. The method of claim 28 said primer pairs and probes of steps a), and d) are combined and amplified together.

34. A kit for determining if a bcr/abl translocation is present in a biological sample comprising at least one oligonucleotide selected from the group consisting of SEQ ID NOs 1-3, 8 and 9.

35. The kit of claim 34 further comprising oligonucleotide for amplifying a housekeeping gene wherein said oligonucleotides comprise either SEQ ID NOs 4-7.

435006/000

edspap.20050037373