Method and device for improved reaction kinetics in nucleic acid hybridizations

A method of detecting the presence of a target polynucleotide comprising:

• a) providing a porous medium capable of transporting by capillarity a solution of single stranded polynucleotides, said medium having a contact site at or near one end and a capture site remote from the contact site, wherein said strip further comprises a first region, said first region being substantially devoid of capture polynucleotide, adjacent said capture site in the direction of said contact site and a second region adjacent said capture site in the opposite direction, and wherein said strip is nonsegmented between said first region and said capture site and between said second region and said capture site;
• b) immobilizing a sample suspected to contain target polynucleotide in single-stranded form at said capture site;
• c) under hybridizing conditions, contacting the contact site with either:
• i) a transport solvent containing a labelled single-stranded polynucleotide complementary to said target polynucleotide;
• or
• ii) a transport solvent capable of mobilizing a labelled single-stranded polynucleotide complementary to said target polynucleotide, said labelled polynucleotide having been previously dried onto a probe site located intermediate the contact site and the remote capture site such that it can be mobilized by transport solvent, the labeled polynucleotide being transported by capillarity to the remote capture site;
• d) prior to or during step c), blocking nonspecific binding sites on said porous medium;
• and
• e) detecting the presence of label, if any, at said remote capture site as a measure of the target polynucleotide.

A method of detecting the presence of a target polynucleotide comprising:

• a) providing a porous medium capable of transporting by capillarity a solution of single stranded polynucleotides, said medium having a contact site at or near one end, and having immobilized at a capture site remote from said contact site, a single-stranded polynucleotide (capture probe) capable of hybridizing to a first portion of a complementary target polynucleotide, wherein said medium further comprises a first region, said first region being substantially devoid of capture polynucleotide, adjacent said capture site in the direction of said contact site and a second region adjacent said capture site in the opposite direction, and wherein said strip is nonsegmented between said first region and said capture site and between said second region and said capture site;
• b) causing a sample suspended to contain target polynucleotide to contact said remote capture site, directly or by solvent transport, under hybridizing conditions to permit hybridization of the capture probe with the first portion of any target polynucleotide present;
• c) during or after step b), contacting the contact site under hybridizing conditions with either:
• i) a transport solvent containing a labelled single-stranded polynucleotide complementary to a second, distinct portion of said target polynucleotide;
• or
• ii) a transport solvent capable of mobilizing a labelled single-stranded polynucleotide complementary to a second distinct portion of said target polynucleotide, said labelled polynucleotide having been previously dried onto a probe site located intermediate the contact site and the remove capture site such that it can be mobilized by transport solvent, the labelled polynucleotide being transported by capillarity to the remote capture site to form a ternary complex of capture probe:target polynucleotide:labelled probe in the presence of target polynucleotide;
• d) prior to or during step c), blocking nonspecific binding sites on said porous medium;
• and
• e) detecting the presence of label, if any, at said remote capture site as a measure of the target polynucleotide.

A method of detecting the presence of a target polynucleotide comprising:

• a) providing a porous medium capable of transporting by capillary a solution of single-stranded polynucleotides, said medium having a contact site at or near one end, and having immobilized at a capture site remote from said contact site, a single-stranded polynucleotide (capture probe) capable of hybridizing to a complementary target polynucleotide, wherein said medium further comprises a first region, said first region being substantially devoid of capture polynucleotide, adjacent said capture site in the direction of said contact site and a second region adjacent said capture site in the opposite direction, and wherein said strip is nonsegmented between said first region and said capture site and between said second region and said capture site;
• b) labelling any target polynucleotide present in a test sample with a detectable label;
• c) contacting the contact site under hybridizing conditions with either;
• i) a transport solvent containing said test sample suspected to contain said labelled target polynucleotide;
• or
• ii) a transport solvent capable of mobilizing a test sample suspected to contain said labelled target polynucleotide, said test sample having been previously dried onto a probe site located intermediate the contact site and the remote capture site such that it can be mobilized by transport solvent, the labelled polynucleotide being transported by capillarity to the remote capture site;
• d) prior to or during step c), blocking nonspecific binding sites on said porous medium;
• and
• e) detecting the presence of label, if any, at said remote capture site as a measure of the target polynucleotide.

In a solid phase hybridization method for detecting the presence of a target polynucleotide involving:

• immobilizing a target polynucleotide, if present in a test sample, directly or via an intermediate capture structure, on a solid phase at a capture site;
• before, during or after said immobilization, attaching a detectable label to said target polynucleotide, if present;
• and detecting said label, if any, at said capture site;
• the improvement comprising:
• a) using a porous chromatographic medium as said solid phase, said medium having a contact site at one end and said capture site being remote from said contact site, wherein said medium further comprises a first region, said first region being substantially devoid of capture polynucleotide, adjacent said capture site in the direction of said contact site and a second region adjacent said capture site in the opposite direction, and wherein said strip is nonsegmented between said first region and said capture site and between said second region and said capture site, and optionally including at least one other site intermediate said contact site and said capture site;
• and
• b) performing at least one of said immobilizing and attaching steps by immobilizing at least one single-stranded polynucleotide in hybridizable form at said capture site and transporting by chromatographic solvent transport from said contact site or said other site at least one single-stranded labelled complementary polynucleotide capable of hybridizing with said immobilized polynucleotide, such that label becomes immobilized at said capture site if target polynucleotide is present;
• wherein at least one hybridization step occurs between an immobilized polynucleotide on said solid phase and a complementary polynucleotide in a mobile phase transported past said immobilized polynucleotide by chromatographic solvent transport.