Method of detecting minor BCR-ABL1 gene

Patent metadata

Field	Value
Publication number	US-12018324-B2
Application number	US-201816607314-A
Country	US
Kind code	B2
Filing date	Apr 25, 2018
Priority date	Apr 26, 2017
Publication date	Jun 25, 2024
Grant date	Jun 25, 2024

Abstract

The disclosure provides a method comprising (1) conducting a PCR using a nucleic acid sample obtained from the subject as the template, a forward primer having a nucleic acid sequence of a part of exon 1 of the BCR gene, and a reverse primer having a nucleic acid sequence complementary to a part of exons 2 to 11 of the ABL1 gene, in the presence of a modified nucleic acid having a nucleic acid sequence of a part of exons 2 to 14 of the BCR gene or a nucleic acid sequence complementary thereto; and (2) determining that the subject has the minor BCR-ABL1 gene when the nucleic acid amplification is occurred in the PCR.

First claim

What is claimed is:

1. A method of detecting the minor BCR-ABL11 gene in a subject, comprising:
(1) conducting a PCR using a nucleic acid sample obtained from the subject as the template, a forward primer having a nucleic acid sequence of a part of exon 1 of the BCR gene, and a reverse primer having a nucleic acid sequence complementary to a part of exons 2 to 11 of the ABL11 gene, and a probe capable of binding to either strand of PCR products derived from the m-BCR-ABL1 mRNA by conducting said PCR, in the presence of a modified nucleic acid having a nucleic acid sequence of a part of exons 2 to 14 of the BCR gene or a nucleic acid sequence complementary thereto,
wherein the modified nucleic acid stops an elongation reaction at the site where said modified nucleic acid is bound and wherein said modified nucleic acid is not degraded by the exonuclease activity of a reverse transcriptase or a DNA polymerase, and
wherein a portion of the nucleic acid sequence of the template complementary to the forward primer and a portion of the nucleic acid sequence of the template complementary to the reverse primer do not overlap a portion of the nucleic acid sequence of the template complementary to the modified nucleic acid; and
(2) determining that the subject has the minor BCR-ABL11 gene when the nucleic acid amplification has occurred in the PCR.

2. The method according to claim 1 , wherein the modified nucleic acid has a nucleic acid sequence of a part of exons 2 to 13 of the BCR gene or a nucleic acid sequence complementary thereto.

3. The method according to claim 1 , wherein the modified nucleic acid has a nucleic acid sequence of a part of exons 12 and 13 of the BCR gene or a nucleic acid sequence complementary thereto.

4. The method according to claim 1 , wherein the modified nucleic acid comprises about 10 to 30 nucleotides.

5. The method according to claim 1 , wherein the modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 8.

6. The method according to claim 1 , wherein the modified nucleic acid comprises a PNA portion.

7. The method according to claim 1 , wherein the nucleic acid sample is cDNA generated by reverse-transcribing an RNA sample obtained from the subject.

8. The method according to claim 1 , wherein the nucleic acid sample is cDNA generated by reverse-transcribing an RNA sample obtained from the subject in the presence of a modified nucleic acid having a nucleic acid sequence complementary to a part of exons 2 to 14 of the BCR gene.

9. The method according to claim 8 , wherein the modified nucleic acid used in the reverse transcription and the modified nucleic acid used in the PCR have the same structure.

10. The method according to claim 7 , wherein the reverse primer used in the reverse transcription and the reverse primer used in the PCR have the same structure.

11. The method according to claim 7 , wherein the reverse transcription and the PCR are carried out in the same container.

12. The method according to claim 1 , wherein the PCR is a quantitative PCR.

13. The method according to claim 1 , wherein the PCR is a quantitative real-time RT-PCR.

14. The method according to claim 1 , wherein the subject is a human being.

15. The method according to claim 1 , wherein the nucleic acid sample is a nucleic acid sample extracted from leukocytes in peripheral blood or nucleated cells in bone marrow aspirate.

16. A method of detecting the expression of the minor BCR-ABL11 gene in a subject, comprising:
(1) reverse-transcribing an RNA sample obtained from the subject using a first reverse primer having a nucleic acid sequence complementary to a part of exons 2 to 11 of the ABL11 gene in the presence of a modified nucleic acid having a nucleic acid sequence complementary to a part of exons 2 to 14 of the BCR gene to obtain a cDNA template,
wherein the modified nucleic acid stops an elongation reaction at the site where said modified nucleic acid is bound and wherein said modified nucleic acid is not degraded by the exonuclease activity of a reverse transcriptase or a DNA polymerase;
(2) conducting a PCR using a forward primer having a nucleic acid sequence of a part of exon 1 of the BCR gene and a second reverse primer having a nucleic acid sequence complementary to a part of exons 2 to 11 of the ABL11 gene, and a probe capable of binding to either strand of the PCR products derived from the m-BCR-ABL1 mRNA by conducting said PCR,
wherein a portion of the nucleic acid sequence of the cDNA template complementary to the forward primer and a portion of the nucleic acid sequence of the cDNA template complementary to the second reverse primer do not overlap a portion of the nucleic acid sequence of the cDNA template complementary to the modified nucleic acid; and
(3) determining that the subject is expressing the minor BCR-ABL11 gene when the nucleic acid amplification has occurred in the PCR.

17. The method according to claim 1 , wherein the forward primer comprises the nucleic acid sequence of SEQ ID NO: 13.

18. The method according to claim 1 , wherein the reverse primer comprises the nucleic acid sequence of SEQ ID NO: 11.

19. The method according to claim 1 , wherein the modified nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 8, the forward primer comprises the nucleic acid sequence of SEQ ID NO: 13, the reverse primer comprises the nucleic acid sequence of SEQ ID NO: 11, and the probe comprises the nucleic acid sequence of SEQ ID NO: 14.

Assignees

• Otsuka Pharma Co Ltd

Inventors

• Katsuragi Kiyonori
• Tanaka Hideaki
• Ito Ryuta
• Koga Daisuke

Classifications

• C12Q2561/113

  Real time assay · CPC title

• C12Q1/6886 Primary

  for cancer (immunoassay for cancer G01N33/575) · CPC title

• C12N15/1096

  cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q1/686 Primary

  Polymerase chain reaction [PCR] · CPC title