Loop probe hybridization assay for polynucleotide analysis

A method of assaying a sample for a first amplification product from a first target polynucleotide, comprising:

• providing a first pair of first and second primers;
• contacting the sample which is suspected of containing the first target polynucleotide with the first primer under conditions in which the first primer can hybridize to the target polynucleotide and can be extended to form a first primer extension product;
• altering the sample conditions to allow dissociation of the first primer extension product from the first target polynucleotide;
• contacting the sample with the second primer under conditions in which the second primer can hybridize to the first primer extension product and be extended to form a second primer extension product, wherein the second primer is complementary at its 3′ end to the first primer extension product at a position in the first primer extension product which is 3′ to the first primer;
• wherein one of the first and second primer extension products thus formed is the first amplification product and comprises a first capture sequence and a first label;
• altering the sample conditions to allow dissociation of the second primer extension product from the first primer extension product;
• contacting the sample with a first probe polynucleotide attached to a substrate under hybridization conditions in which the first probe polynucleotide can hybridize to the first amplification product;
• wherein the first probe polynucleotide comprises first and second complementary regions and a third region located between the first and second complementary regions, and further wherein the first probe polynucleotide can form a stem-loop structure in which the first and second complementary regions hybridize to each other to form a stem and the third region forms a loop;
• wherein at least a part of the third region is complementary to at least a part of the first capture sequence, and wherein the first probe polynucleotide preferentially hybridizes to the first amplification product to thereby disrupt formation of the stem-loop structure under the hybridization conditions;
• and
• determining if the first label is associated with the first probe polynucleotide.

A method of forming an amplification product assay complex, comprising:

• hybridizing the amplification product to an unlabelled probe polynucleotide attached to a substrate under a first set of hybridization conditions to form an amplification product assay complex;
• wherein the amplification product comprises a first label and a first single-stranded capture sequence;
• wherein the probe polynucleotide comprises first and second complementary regions and a third region located between the first and second complementary regions, and further wherein the probe polynucleotide can form a stem-loop structure in which the first and second complementary regions hybridize to each other to form a stem and the third region forms a loop;
• wherein at least a part of the third region is complementary to at least a part of the capture sequence, and wherein the probe polynucleotide hybridizes to the amplification product and thereby disrupts formation of the stem-loop structure under the first set of hybridization conditions.

A kit comprising:

• a substrate attached to an unlabeled probe polynucleotide comprising first and second complementary regions and a third region located between the first and second complementary regions, wherein the probe polynucleotide can form a stem-loop structure in which the first and second complementary regions hybridize to each other to form a stem and the third region forms a loop, wherein at least a part of the third region is complementary to at least a part of a capture sequence of an amplification product from a target polynucleotide, wherein the unlabeled probe polynucleotide can preferentially hybridize to the amplification product and thereby disrupt formation of the stem-loop structure under at least one set of hybridization conditions;
• a reagent for incorporating a label into the amplification product;
• a housing for retaining the substrate and the reagent;
• and
• instructions provided with said housing that describe how to use the components of the kit to assay a sample for the amplification product.