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US 6,057,099 AGranted
Detection of nucleic acid sequences
AssigneeIntelligene Ltd.
InventorsAsher Nathan, Yaron Tikochinski, Ed Rudd, Guido Krupp
Life of this patent
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Abstract
A method for detecting the presence of an assayed nucleic acid sequence in a sample is an essentially two stage-procedure. As illustrated, in a first stage the sample is reacted in a manner which gives rise to the production of a triggering oligonucleotide where the sample contains the assayed sequence. In the second stage, the reaction product is incubated under appropriate conditions with an amplification system whereby, in the presence of triggering oligonucleotide, a large amount of a nucleic acid product is obtained. The detection of this product thus indicates the presence of the assayed sequence in the sample.
Claims20
A method for detecting the presence of an assayed nucleic acid sequence in a sample, comprising the steps of:
- (a) incubating the sample within a detection system comprising:
- (i) a first oligonucleotide having a double-stranded promoter sequence and a 5' end sequence which is complementary to the 5' end portion of the assayed nucleic acid sequence;
- (ii) a second oligonucleotide having a single-stranded 3' end sequence being complementary to a 3' end portion of the assayed nucleic acid sequence, and further having a sequence which can be transcribed into a triggering oligonucleotide capable of initiating a reaction in a transcription system in which a transcription product is produced;
- the 3' end sequence of the second oligonucleotide and the 5' end sequence of the first oligonucleotide are either complementary together to the entire assayed nucleic acid sequence or to only part thereof, thus leaving an intermediate portion in the assayed nucleic acid sequence having no complementary counterpart in either the first or the second oligonucleotide, in which latter case the detection ensemble further comprises
- (iii) a third oligonucleotide being complementary to said intermediate portion;
- said first, second and third oligonucleotides, being either DNA oligonucleotides, a DNA oligonucleotide comprising RNA nucleotides (rNTPs), or a DNA oligonucleotide comprising analogs of DNA nucleotides (dNTPs) or of rNTPs;
- (b) providing conditions allowing hybridization of said first oligonucleotide and said second oligonucleotide, and where present also said third oligonucleotide, with said assayed nucleic acid sequence, and optionally providing also a ligase to allow ligation of adjacent ends of said first, second and third oligonucleotides;
- (c) providing conditions allowing the production of said triggering oligonucleotide, said conditions comprising the provision of:
- (i) an RNA polymerase;
- (ii) 4 species of nucleotides, of which at least two are rNTPs and of the remaining two
- at least one is a non-rNTP being a dNTP or a nucleotide analog other than rNTP or dNTP, or
- at least one is a combination of rNTP and said non-rNTP of the same nucleotide species, the said non-rNTP being in abundance over said rNTP;
- and
- (iii) conditions allowing the incorporation of rNTPs into the triggering oligonucleotide produced by the RNA polymerase;
- (d) contacting the triggering oligonucleotide with an amplification system in which the triggering oligonucleotide induces formation of reporter oligonucleotides;
- and
- (e) detecting the presence of said reporter oligonucleotide, positive results indicating the presence of said assayed nucleic acid sequence, in said sample.
14 more elements
A method for detecting the presence of an assayed nucleic acid sequence in a sample, comprising the step of:
- (a) reacting the sample with a detection system comprising:
- a single-stranded first oligonucleotide having from its 3' to its 5' end three sequence portions linked to one another comprising a first arbitrary portion, a second arbitrary portion of 1-5 bases long, and a 5' end sequence which is complementary to the 5' end portion of the assayed nucleic acid sequence;
- a second oligonucleotide comprising a single-stranded 3' end sequence being complementary to a 3' end portion of the assayed nucleic acid sequence, and further comprising a third double-stranded arbitrary portion;
- (b) incubating under conditions to allow hybridization of the assayed nucleic acid sequence to said first single-stranded oligonucleotide and to said second oligonucleotide to produce oligonucleotide hybrids;
- (c) raising the temperature to a point in which only essentially perfectly matched hybrids formed in step (b) remain hybridized while all other hybrids in which the individual strands do not perfectly match to one another are melted;
- (d) adding a blocker oligonucleotide molecule, being a single-stranded oligonucleotide comprising at its 5' end a sequence complementary to said second arbitrary portion and a sequence, at its 3' end complementary to the 5' end portion of the assayed nucleic acid sequence and providing conditions allowing hybridization of said blocker molecule with free said first single-stranded oligonucleotide;
- adding a promoter DNA molecule, comprising a double-stranded functional promoter and a sequence complementary to the first and second arbitrary portions attached to a non-template strand and providing conditions for hybridization, whereby a hybrid comprising the assayed nucleic acid sequence, said promoter oligonucleotide molecule, said first single-stranded oligonucleotide and said second oligonucleotide is obtained from which an oligonucleotide transcript can be transcribed;
- (e) adding transcription reagents and incubating under conditions to allow the formation of oligonucleotide transcripts having a triggering oligonucleotide;
- (f) contacting the oligonucleotide transcripts with an amplification system in which the triggering oligonucleotide sequence induces formation of oligonucleotide molecules containing the reporter nucleotide sequence;
- and
- (g) detecting the presence of said reporter oligonucleotide sequence, positive results indicating the presence of said assayed oligonucleotide sequence in said sample.
8 more elements
Dependencies are inferred from the claim text.