Methods and compositions for sequence-specific purification and multiplex analysis of nucleic acids
AssigneeQiagen Gaithersburg, Inc.
InventorsDominic O'neill, Irina Nazarenko, Holly Basham, Arvind Virmani, Shiuli Agarwal
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Abstract
Methods and materials for determining the presence of at least one nucleic acid in a sample are provided, said methods comprising (1) a purification step using sequence specific hybrid capture; (2) an amplification step; and (3) a detection step using two separate sequence-specific polynucleotide probes. Also provided are nucleic acids comprising SEQ ID NO: 1 to SEQ ID NO: 727 and nucleic acid probes and probe sets comprising the same.
Claims43
An isolated nucleic acid having an overall length of not more than 100 nucleotides comprising at least one nucleotide sequence having at least 75-percent homology to a nucleotide sequence selected from the group consisting of SEQ ID NO:
- 1 to SEQ ID NO:
- 727 and complements thereof.
A method of detecting at least one target nucleic acid in a sample comprising non-target nucleic acids, said method comprising:
- a. purifying the target nucleic acid from the sample by a method comprising:
- (i) contacting the sample with at least one purification probe, wherein at least a portion of the nucleic acid probe hybridizes to the at least one target nucleic acid to form a DNA:RNA hybrid;
- (ii) immobilizing the DNA:RNA hybrid to a first solid support by a method comprising contacting the DNA:RNA hybrid with at least a first antibody capable of binding to the DNA:RNA hybrid, wherein the antibody is bound to or adapted to be bound to the first solid support;
- and
- (iii) separating the first solid support from the sample to generate at least one purified target nucleic acid;
- b. genotyping the purified target nucleic acid by a method comprising:
- (i) amplifying at least a portion of the purified target nucleic acid to generate an amplicon;
- (ii) immobilizing the amplicon to a second solid support by a method comprising contacting the amplicon with at least one immobilization probe, wherein:
- (α) the immobilization probe is bound to or adapted to be bound to the second solid support;
- and
- (β) at least a portion of the immobilization probe hybridizes the at least one target nucleic acid;
- (iii) contacting the immobilized amplicon with at least one detection probe which is optionally biotinylated, wherein the at least a portion of the detection probe hybridizes to the at least one target nucleic acid to generate a detection complex;
- and
- (iv) detecting at least a first detectable signal generated by the detection complex, wherein the detectable signal indicates the genotype of the target nucleic acid.
Dependencies are inferred from the claim text.