Nucleic acid probes for in situ hybridization

A method for detecting a preselected nucleic acid target sequence in a sample, wherein the sample is an in situ preparation of tissue or collected cells;

• the method comprising:
• (a) providing a hybridization probe comprising a series of first nucleic acid segments consecutively complementary to the preselected nucleic acid target sequence, and between each adjacent pair of first nucleic acid segments, a spacer nucleic acid segment, wherein the spacer segments are identical or different and are not substantially complementary to the preselected nucleic acid target sequence, and further wherein the spacer segments are not substantially complementary to the first segments;
• wherein the hybridization probe is labeled;
• (b) contacting the hybridization probe to the sample comprising the preselected nucleic acid target sequence under conditions such that the hybridization probe hybridizes to the preselected nucleic acid target sequence;
• (c) removing excess hybridization probe that is not hybridized to the preselected nucleic acid target sequence;
• and
• (d) detecting the label on the hybridization probe.

A method for detecting a preselected nucleic acid target sequence in a sample, the method comprising:

• (a) providing a hybridization probe comprising a series of first nucleic acid segments consecutively complementary to the preselected nucleic acid target sequence, and between each adjacent pair of first nucleic acid segments, a spacer nucleic acid segment, wherein the spacer segments are identical or different and are not substantially complementary to the preselected nucleic acid target sequence, and further wherein the spacer segments are not substantially complementary to the first segments;
• (b) contacting the hybridization probe to the sample under conditions such that the hybridization probe hybridizes to the preselected nucleic acid target sequence;
• (c) contacting the sample of (b) with a detection probe that is complementary to the one or more spacer segments of the hybridization probe, wherein the detection probe comprises one or more nucleic acids with detectable labels and wherein the detection probe hybridizes to the hybridization probe;
• and
• (d) detecting the presence of the preselected nucleic acid target based on the presence of the detection probe.

A composition of nucleic acid hybridization probes that hybridize to distinct segments of a preselected nucleic acid target sequence wherein each probe consists of

• a first nucleic acid segment that is complementary to a segment of the preselected nucleic acid target sequence and directly attached to a spacer segment that is not substantially complementary to the preselected nucleic acid target sequence or to the first nucleic acid segment,
• wherein the spacer segment is incorporated into the probe at the 3′ end of the first nucleic acid segment;
• and
• wherein a chemically introduced hapten molecule and a chemically introduced fluorescent moiety are integrated into the spacer segment.

A method for preparing a nucleic acid hybridization probe composition for detecting a preselected nucleic acid target sequence, the method comprising

• chemically reacting ribonucleoside triphosphates or deoxynucleoside triphosphates and labeled phosphoramidites in an amine-exchange reaction to form the hybridization probe,
• wherein the labeled phosphoramidites comprise a phosphoramidite selected from the group consisting of cyanine 3 phosphoramidite, cyanine 5 phosphoramidite, fluorescein phosphoramidite, 6-fluorescein phosphoramidite, and 6-hexachloro-fluorescein phosphoramidite;
• the hybridization probe comprises a series of first nucleic acid segments consecutively complementary to the preselected nucleic acid target sequence, and between each adjacent pair of first nucleic acid segments, a spacer nucleic acid segment, wherein the spacer segments are identical or different and are not substantially complementary to the preselected nucleic acid target sequence, and the spacer segments are not substantially complementary to the first segments;
• and the labeled phosphoramidites are present in the spacer segments.

A method for preparing a nucleic acid hybridization probe composition for detection of a nucleic acid target sequence, comprising the steps of:

• preselecting the nucleic acid target sequence;
• providing an in vitro mixture of
• (a) a DNA construct comprising an RNA promoter operably linked to a template DNA sequence, wherein the template DNA sequence encodes a non-naturally occurring linear nucleic acid molecule having a 5′ end and a 3′ end and comprising
• a series of first nucleic acid segments consecutively complementary to the preselected nucleic acid target sequence, and
• between each adjacent pair of the first nucleic acid segments, a spacer nucleic acid segment,
• wherein the spacer nucleic acid segments between each adjacent pair of the first nucleic acid segments are either the same or different,
• wherein the spacer nucleic acid segments are not substantially complementary to any part of the preselected nucleic acid target sequence, and
• wherein the spacer nucleic acid segments are not substantially complementary to any part of the first nucleic acid segments;
• wherein at least one spacer nucleic acid segment comprises a subsequence that renders the at least one spacer nucleic acid segment a target for a polynucleic acid cleaving enzyme in the presence of 0 or more accessory molecule(s) required for the cleaving enzyme to cleave a substrate;
• (b) a mixture of different ribonucleoside triphosphates wherein at least some of said ribonucleoside triphosphates are chemically labeled;
• (c) an RNA polymerase capable of transcribing an RNA molecule comprising residues of the chemically labeled ribonucleoside triphosphates from the template DNA sequence under control of the RNA promoter;
• incubating said mixture under conditions permissive for transcription of the RNA molecule by the RNA polymerase, wherein said RNA molecule is thereby transcribed in quantity;
• incubating the resulting RNA molecule with the polynucleic acid cleaving enzyme in the presence of 0 or more accessory molecule(s) to sequence specifically cleave at least some of the spacer nucleic acid segments of the quantity of RNA molecule to obtain a fragmented nucleic acid hybridization probe composition.