Method for increasing the sensitivity of nucleic acid hybridization assays

• US 4,824,776 A
• Filed: 07/25/1985
• Issued: 04/25/1989
• Est. Priority Date: 07/25/1985
• Status: Expired due to Term

• Current Assignee: Syngene Incorporated
• Original Assignee: Molecular Biosystems, Inc (Alliance Pharmaceutical Corporation)
• Inventors: Heller, Michael J.
• Primary Examiner(s): Kepplinger, Esther M.
• Assistant Examiner(s): Wagner, Richard
• Application Number:
  US06/759,047
• Time in Patent Office: 1,370 Days
• Field of Search: 435/6, 435/803, 436/501, 436/518, 436/531, 536/27, 935/2, 935/8, 935/9, 935/10, 935/78
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/705 for herpetoviridae, e.g. herpes simplex, varicella zoster
• CPC Class Codes: Y10S 435/803 Physical recovery methods, e.g. chromatography, grinding

Abstract

An improvement in the sensitivity of hybridization assays for detecting polynucleotide target sequences is provided by selective dehybridization of the bound portion of the probe, separation of the dehybridized portion from the support and the residual sample, and concentrating the separated probe before detecting its presence by means of its reporter group. The concentration step can be carried out by adsorption of the dehybridized probe onto a basic ion exchange material. A displacer sequence having greater binding affinity for the target polynucleotide sequence may be used to dehybridize the bound probe portion for subsequent concentration.

Claims

1. The hybridization assay method for detecting a specific polynucleotide target sequence wherein a nucleic acid containing target sample in single stranded form is affixed to a support, contacted thereon under hybridizing conditions with a non-radioactive reporter group labeled single-stranded polynucleotide probe having a sequence complementary to the target sequence, said probe being bound to said affixed sample when the target is present, said probe hybridizing with said target sequence, then removing the unbound portion of said probe from said support, and detecting the presence of said probe by means of its reporter group, wherein the sensitivity of the assay is increased by employing the additional steps comprising:

(a) after removal of the unbound portion of the probe, dehybridizing the bound portion;

(b) concentrating the dehybridized separated probe onto a solid support; and

(c) detecting the presence of said probe on said solid support by means of said non-radioactive reporter group.

2. The method of claim 1 in which the dehybridized portion of the probe is concentrated by adsorption from an aqueous solution onto a basic anion exchange material binding said probe.

3. The method of claim 2 in which the anion exchanger functional groups are quaternary amino groups.

4. The method of claim 3 in which said anion exchange functional groups are provided on a substrate selected from cross-linked dextran or glass.

5. The method of claim 1 in which the bound labelled probe portion is dehybridized by contacting the supported sample with a displacer probe comprising a single stranded polynucleotide sequence longer than the labelled probe including the same sequence as the labelled probe and an additional sequence complementary to the sequence of said target sample adjacent said target sequence to which the labelled probe is bound, said additional sequence being of sufficient length to cause said displacer to preferentially bind to said target sequence and subsequently displace said labelled probe therefrom.

6. A hybridization assay method for detecting a specific target sequence wherein a nucleic acid sample in single-stranded form is affixed to a support, contacted thereon with a non-radioactively labelled polynucleotide probe having a single-stranded sequence complementary to the target sequence to be directed, said probe comprising a sequence of 10 to 50 nucleotide units and having a reporter group attached thereto, said probe being bound to said affixed sample when the specific target is present, removing the unbound portion of said probe from said support, and detecting the presence of said probe by means of its reporter group, wherein the sensitivity to the assay is increased by employing the additional steps comprising:

(a) after removal of the unbound portion of the probe, dehybridizing the bound portion and forming an aqueous solution thereof;

(b) contacting the separated solution with an anion exchange material for binding said probe so as to concentrate said probe onto the anion exchange material; and

(c) detecting its presence on said anion exchange material by means of said non-radioactive reporter groups.

7. The method of claim 6 in which said anion exchange material comprises an adsorbent substrate selected from cross-linked dextran or glass with substituted amino groups attached thereto.

8. The method of claim 6 in which said probe is labelled with a fluorescent reporter group.

9. The method of claim 6 in which the bound probe portion is dehybridized by contacting the supported sample with a displacer probe comprising a single-stranded polynucleotide sequence longer than the labelled probe including the same sequence as the probe and an additional sequence complementary to the sequence of said target sample adjacent said target sequence to which the probe is bound, said additional sequence being of sufficient length to cause said displacer to preferentially bind to said target sequence and displace said probe therefrom.

10. The hybridization assay method for detecting a specific polynucleotide target sequence wherein a nucleic acid sample containing target in single-stranded form is affixed to a support, contacted thereon under hybridizing conditions with a polynucleotide probe having a single-stranded sequence complementary to a characterizing target sequence to be detected, said probe comprising a sequence of from 10 to 50 nucleotide units and having a fluorescent label attached thereto, probe being bound to said affixed sample when the specific target is present, said complementary sequence of the probe hybridizing with said target sequence, removing the unbound portion of said portion of said probe from said support, and detecting the presence of said probe by means of its reporter group, wherein the sensitivity of the assay is increased by employing the additional steps comprising:

(a) after removal of the unbound portion of the probe, dehybridizing the bound portion of the probe and forming an aqueous solution thereof;

(b) contacting the separated solution with an anion exchange material comprising a substrate selected from cross-linked dextran or glass with substituted amino group provided thereon so as to concentrate the probe on said anion exchange material; and

(c) detecting the presence of said probe on said anion exchange material by means of said fluorescent label.

11. The method of claim 10 in which said anion exchange material is in the form of porous generally spherical beads.

12. The method of claim 10 in which the bound labelled probe portion is dehybridized by contacting the supported sample with a displacer comprising a single stranded polynucleotide sequence longer than the labelled probe including the same sequence as the labelled probe and an additional sequence complementary to the sequence of said target sample adjacent said target sequence to which the probe is bound, said additional sequence being of sufficient length to cause said displacer to preferentially bind to said target sequence and displace said probe therefrom.

13. The method of claim 10 in which said anion exchange material comprises porous beads of cross-linked dextran having substituted amino groups attached thereto.

14. The method of claim 10 in which said anion exchange material comprises porous beads of silica glass having substituted amino groups attached thereto.