Nucleic acid hybridization technique and kit therefor

Details Nucleic acid hybridization technique and kit therefor Nucleic acid hybridization technique and kit therefor

C - Chemistry, Metallurgy

12

Q

150/8.5

C12Q 1/68 (2006.01)

Patent

CA 1309932

5191/98D-106 ABSTRACT A novel assay method is suitable for detecting a polynucleotide in a liquid sample. The method comprises the steps of: (a) combining the sample with at least two different probes, being a first probe and a second probe, to form a reaction mixture, each probe comprising a single- stranded polynucleotide which contains a nucleotide sequence complementary to a portion of the target polynucleotide, the probes together forming hybrid molecules by complementary base pairing with the target polynucleotide, neither probe being affixed to a solid carrier, the probes do not hybridize with each other, and the second probe having a detectable label; (b) subsequently contacting the reaction mixture with a solid carrier which binds the first probe but not the second probe and not the target polynucleotide; and (c) subsequently determining if any of the label is bound on the solid carrier. The method combines the speed and reaction efficiency of solution hybridization assays, and ease of separation of solid carrier hybridization assays. A kit for use with the above method is also disclosed. The method and kit of the present invention are also suitable for diagnosis of genetic diseases, e.g. sickle cell anemia, and for cancer diagnosis. For example, the assay method can be used to detect a target polynucleotide in a sample which may also contain a standard 5191/98D-106 polynucleotide, the nucleotide sequences of the two polynucleotides being substantially similar, the standard polynucleotide having a portion A and a portion B, the target polynucleotide having a portion A' and a portion B', the standard and target polynucleotides differing in at least one nucleotide which is located between portions A and B on the standard polynucleotide and between portions A' and B' on the target polynucleotide, the method comprising the steps of: (a) severing both the standard and target polynucleotides present into single-stranded segments, such that none of the segments of the standard polynucleotide contains both portions A and B, while at least one of the segments of the target polynucleotide contains both portions A' and B'; (b) combining the treated sample from step (a) with at least two probes, being a first probe and a second probe, to form a reaction mixture, each probe comprising a single-stranded polynucleotide, the probes being unable to hybridize with each other, the first probe complementary base pairing with portion A, and with portion A', the second probe complementary base pairing with portion B, and with portion B', the two probes together forming hybrid molecules by complementary base pairing with the single-stranded segment of the target polynucleotide which contains both portions A' and B'; and (c) subsequently determining the presence of hybrid molecules containing both of the probes.

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