Method for detecting chromosomal rearrangements
First Claim
1. An in vitro method for detecting chromosomal rearrangements between at least two specific chromosomal regions in a biological sample of a human subject, wherein said rearrangement is a ELM4-ALK, ROS1-CD74, ROS1-EZR, or ROS1-SLC34A2 chromosomal rearrangement, said method comprising the steps of:
- a) isolating deoxyribonucleic acid (DNA) molecules comprising said specific chromosomal regions from said biological sample, wherein said DNA molecules have an average length of X base pairs;
b) amplifying the DNA molecules of step a) by a multiplex polymerase chain reaction assay,said assay comprising at least two sets of primers, wherein each set of primers is capable of hybridizing with a specific chromosomal region, andeach set of primer comprises a plurality of primers,said primers being capable of hybridizing to a nucleic acid strand of one of the said specific chromosomal regions at sites regularly spaced of less than X/2 base pairs; and
c) hybridizing the product of the amplification of step b) with at least one set of nucleic probes,wherein said set of nucleic acid probes comprises a plurality of nucleic acid probes,said probes being capable of hybridizing to a nucleic acid strand of one of the said specific chromosomal regions at sites regularly spaced of less than X/2 base pairs; and
the 5′
end of said probe being capable of hybridizing within 0 to 30 bases from the 3′
end of a primer capable of hybridizing to the same specific chromosomal region; and
wherein the successful hybridization of at least one probe of said set of nucleic probes to product of the amplification of step b) indicates the presence of a chromosomal rearrangement.
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Abstract
An object of the invention is an in vitro method for detecting chromosomal rearrangements between at least two specific chromosomal regions in a biological sample of a human subject, which comprises the steps of: a) isolating deoxyribonucleic acid (DNA) molecules comprising said specific chromosomal regions from said biological sample, wherein said DNA molecules have an average length of X base pairs; b)amplifying the DNA molecules of step a) by a multiplex polymerase chain reaction assay, said assay comprising at least two sets of primers, wherein each set of primers is capable of hybridizing with a specific reference chromosomal region, and each set of primer comprises a plurality of primers, said primers being capable of hybridizing to a nucleic acid strand of one of the said specific chromosomal regions at sites regularly spaced of less than X/2 base pairs; and hybridizing the product of the amplification of step b) with at least one set of nucleic probes.
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19 Claims
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1. An in vitro method for detecting chromosomal rearrangements between at least two specific chromosomal regions in a biological sample of a human subject, wherein said rearrangement is a ELM4-ALK, ROS1-CD74, ROS1-EZR, or ROS1-SLC34A2 chromosomal rearrangement, said method comprising the steps of:
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a) isolating deoxyribonucleic acid (DNA) molecules comprising said specific chromosomal regions from said biological sample, wherein said DNA molecules have an average length of X base pairs; b) amplifying the DNA molecules of step a) by a multiplex polymerase chain reaction assay, said assay comprising at least two sets of primers, wherein each set of primers is capable of hybridizing with a specific chromosomal region, and each set of primer comprises a plurality of primers, said primers being capable of hybridizing to a nucleic acid strand of one of the said specific chromosomal regions at sites regularly spaced of less than X/2 base pairs; and c) hybridizing the product of the amplification of step b) with at least one set of nucleic probes, wherein said set of nucleic acid probes comprises a plurality of nucleic acid probes, said probes being capable of hybridizing to a nucleic acid strand of one of the said specific chromosomal regions at sites regularly spaced of less than X/2 base pairs; and the 5′
end of said probe being capable of hybridizing within 0 to 30 bases from the 3′
end of a primer capable of hybridizing to the same specific chromosomal region; andwherein the successful hybridization of at least one probe of said set of nucleic probes to product of the amplification of step b) indicates the presence of a chromosomal rearrangement. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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Specification