Chimeric messenger RNA detection methods
First Claim
Patent Images
1. A method for detecting a chimeric mRNA in a biological sample, wherein said chimeric mRNA is associated with acute lymphocytic leukemia (ALL) and comprises a BCR exon in junction with an ABL exon, the method has the step of:
- a) synthesizing cDNA from mRNA in said sample;
b) contacting said cDNA with a first and second primer, wherein said first primer is homologous to a sequence contained in said BCR exon and said second primer is complementary to a sequence in said ABL exon to provide a mixture for amplifying chimeric mRNA associated with ALL;
c) treating the mixture prepared in step (b) under conditions and with reagents suitable for amplifying said cDNA segment by a polymerase chain reaction; and
d) determining if amplification has occurred.
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Abstract
The invention provides highly sensitive methods for detecting specific sequences contained in chimeric mRNA. The mRNA sequences are reverse transcribed into complementary DNA (cDNA), amplified by the Polymerase Chain Reaction, and detected by hybridization with a labeled sequence specific oligonucleotide probe. The method is particularly valuable for the detection of chimeric mRNAs experessed by activated oncogenes that result from aberrant genetic rearragements such as chromosomal translocations.
100 Citations
24 Claims
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1. A method for detecting a chimeric mRNA in a biological sample, wherein said chimeric mRNA is associated with acute lymphocytic leukemia (ALL) and comprises a BCR exon in junction with an ABL exon, the method has the step of:
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a) synthesizing cDNA from mRNA in said sample; b) contacting said cDNA with a first and second primer, wherein said first primer is homologous to a sequence contained in said BCR exon and said second primer is complementary to a sequence in said ABL exon to provide a mixture for amplifying chimeric mRNA associated with ALL; c) treating the mixture prepared in step (b) under conditions and with reagents suitable for amplifying said cDNA segment by a polymerase chain reaction; and d) determining if amplification has occurred. - View Dependent Claims (2, 3, 4)
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5. A method for detecting a chimeric mRNA in a biological sample, wherein said chimeric mRNA is associated with chronic myeloid leukemia (CML) and comprises a BCR exon in junction with an ABL exon, the method has the steps of:
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a) synthesizing cDNA from mRNA in said sample; b) contacting said cDNA with a first and second primer, wherein said first primer has the sequence;
space="preserve" listing-type="equation">5'"'"'GGAGCTGCAGATGCTGACCAAC3'"'"',and said second primer has the sequence;
space="preserve" listing-type="equation">5'"'"'TCAGACCCTGAGGCTCAAAGTC3'"'"',to provide a mixture for amplifying chimeric mRNA associated with CML; c) treating the mixture prepared in step (b) under conditions and with reagents suitable for amplifying said cDNA segment by a polymerase chain reaction; and d) determining if amplification has occurred. - View Dependent Claims (6, 7)
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8. A method for distinguishing between chimeric mRNA associated with chronic myeloid leukemia (CML) and chimeric mRNA associated with acute lymphocytic leukemia (ALL), in a biological sample, wherein said chimeric mRNAs each comprise a BCR exon in junction with an ABL exon, the method has the steps of:
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a) synthesizing cDNA from mRNA in said sample; b) contacting said cDNA with a first and second primer for amplifying chimeric mRNA associated with CML that contains an exon-junction between a BCR exon and an ABL exon; c) treating the mixture prepared in step (b) under conditions and with reagents suitable for amplifying said cDNA segment by a polymerase chain reaction; and d) contacting said cDNA of step (a) with a third and fourth primer, for amplifying chimeric mRNA associated with ALL that contains an exon-junction between a BCR exon and an ABL exon; e) treating the mixture prepared in step (d) under conditions and with reagents suitable for amplifying said cDNA segment by a polymerase chain reaction; and f) determining if amplification has occurred in each of step (c) and step (e). - View Dependent Claims (9, 10, 11, 12)
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13. A kit for detecting a chimeric mRNA associated with chronic myeloid leukemia (CML), wherein said chimeric mRNA comprises a BCR exon in junction with an ABL exon, said kit comprising:
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a) a first and second primer wherein said first primer has the sequence;
space="preserve" listing-type="equation">5'"'"'GGAGCTGCAGATGCTGACCAAC3'"'"',and said second primer has the sequence;
space="preserve" listing-type="equation">5'"'"'TCAGACCCTGAGGCTCAAAGTC3'"'"',b) an oligonucleotide probe. - View Dependent Claims (14, 15)
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16. A kit for detecting a chimeric mRNA associated with acute lymphocytic leukemia, wherein said chimeric mRNA comprises a BCR exon in junction with an ABL exon, said kit comprising:
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a) a first and second primer where said first primer is homologous to a sequence contained in said BCR exon and said second primer is complementary to a sequence in said ABL exon; and b) an oligonucleotide probe specific for the exon junction between said BCR exon and said ABL exon. - View Dependent Claims (17, 18)
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19. A kit for distinguishing between chimeric mRNA associated with chronic myeloid leukemia (CML) and chimeric mRNA associated with acute lymphocytic leukemia (ALL), in a biological sample, wherein said chimeric mRNAs each comprise a BCR exon in junction with an ABL exon, said kit comprising:
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a) a first and second primer for amplifying chimeric mRNA associated with CML which contains an exon-junction between a BCR exon and an ABL exon; b) a third and fourth primer for amplifying chimeric mRNA associated with ALL which contains an exon-junction between a BCR exon and an ABL exon; c) a first oligonucleotide probe specific for said chimeric mRNA associated with CML; and d) a second oligonucleotide probe specific for said chimeric mRNA associated with ALL. - View Dependent Claims (20, 21, 22, 23, 24)
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21. The kit of claim 19 that further comprises reagents for carrying out polymerase chain reaction.
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22. The kit of claim 19, wherein said first probe has the sequence:
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space="preserve" listing-type="equation">5'"'"'GCTGAAGGGCTTTTGAACTCTGCTTA3'"'"';and said second probe has the sequence;
space="preserve" listing-type="equation">5'"'"'GCTGAAGGGCTTCTGCGTCTCCAT3'"'"'.
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23. The kit of claim 19 that further comprises a third oligonucleotide probe, wherein said third probe is distinct from said first oligonucleotide probe and is specific for said chimeric mRNA associated with chronic myeloid leukemia.
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24. The kit of claim 23 wherein said third probe has the sequence:
space="preserve" listing-type="equation">5'"'"'GCTGAAGGGCTTCTTCCTTATTGATG3'"'"'.
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Specification