Method and kit for nucleic acid sequence detection
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Publication Number
US-8673567-B2
Publication Date
2014-03-18
Expiration Date
Abstract
A method and kit for detecting the presence of a target sequence in a polynucleotide analyte contained in a sample are disclosed. In practicing the method, the sample is mixed with a single-stranded DNA target probe having a sequence capable of hybridizing with the target sequence, under conditions effective to form a double-stranded complex of the analyte and the single-stranded DNA target probe, and the single-stranded DNA target probe in the complex is reacted in the presence of a polymerase and one to three nucleotide triphosphates, to add a selected one or more target-directed nucleotide bases to single-stranded DNA target probe's 3′ end to produce a modified probe. The modified probe is hybridized with a single-stranded DNA detection probe, the two probes are ligated to form a two-probe ligation product, and the presence of the ligation product is detected.
Core Innovation
The invention provides a nucleic-acid detection method in which an unamplified polynucleotide analyte in a sample is contacted with a single-stranded DNA target probe having a sequence capable of hybridizing with a target sequence in the polynucleotide analyte to form a double-stranded complex. The single-stranded DNA target probe has a hairpin structure, and a polymerase incubates the double-stranded complex in the presence of no more than three kinds of nucleotide triphosphates, adding selected target-directed nucleotide bases to the 3′ end of the single-stranded DNA target probe.
The polymerase-controlled addition produces a modified single-stranded DNA target probe having a hairpin structure and a 3′ sticky end. The modified single-stranded DNA target probe is then hybridized with a single-stranded DNA detection probe having a hairpin structure with a 3′ sticky end complementary to the 3′ sticky end of the modified single-stranded DNA target probe. The detection probe is defined to not hybridize to the target sequence in the polynucleotide analyte and not ligate with the unmodified single-stranded DNA target probe.
The modified single-stranded DNA target probe and the single-stranded DNA detection probe are ligated to form a circular two-probe ligation product, where the polynucleotide analyte is not used as a template to form the circular two-probe ligation product. After formation of the circular two-probe ligation product, linear nucleic acid molecules are degraded and the circular two-probe ligation product is amplified to produce a detectable amplicon, which is detected by detecting the amplicon.
Claims Coverage
The partial content includes three independent claims. Across these independent claims, three core inventive feature sets are presented: hairpin-structured target and detection probes ligated into a circular two-probe ligation product without using the analyte as a template, extension of the target probe using a polymerase with no more than three kinds of nucleotide triphosphates to create a 3′ sticky end, and post-processing and detection including degradation, amplification to a detectable amplicon, and detection of the amplicon.
Hairpin-structured target probe forming a double-stranded complex with an unamplified polynucleotide analyte
Contacting a sample containing an unamplified polynucleotide analyte with a single-stranded DNA target probe having a sequence capable of hybridizing with a target sequence in the polynucleotide analyte under conditions effective to form a double-stranded complex, wherein the single-stranded DNA target probe has a hairpin structure.
Polymerase extension with no more than three kinds of nucleotide triphosphates to generate a 3′ sticky end
Incubating the double-stranded complex in the presence of a polymerase and no more than three kinds of nucleotide triphosphates to add selected one or more target-directed nucleotide bases to the single-stranded DNA target probe’s 3′ end to produce a modified single-stranded DNA target probe having a hairpin structure and a 3′ sticky end.
Hairpin detection probe hybridization and ligation into a circular two-probe ligation product without template use of the polynucleotide analyte
Hybridizing the modified single-stranded DNA target probe with a single-stranded DNA detection probe that has a hairpin structure with a 3′ sticky end complementary to the 3′ sticky end of the modified single-stranded DNA target probe, wherein the detection probe does not hybridize to the target sequence in the polynucleotide analyte, does not ligate with the unmodified single-stranded DNA target probe, and ligating the modified target probe and the detection probe forms a circular two-probe ligation product, wherein the polynucleotide analyte is not used as a template to form the circular two-probe ligation product.
Post-ligation degradation, amplification to a detectable amplicon, and amplicon detection
After formation of the circular two-probe ligation product, degrading linear nucleic acid molecules, amplifying the circular two-probe ligation product to produce a detectable amplicon, and detecting the presence of the circular two-probe ligation product by detecting the amplicon.
Attaching a polynucleotide target sequence to a non-polynucleotide analyte for hairpin-based circular two-probe ligation detection
Attaching a polynucleotide target sequence to the non-polynucleotide analyte and detecting the attached polynucleotide target sequence by contacting the attached polynucleotide target sequence with a hairpin-structured single-stranded DNA target probe, incubating in the presence of a polymerase and no more than three kinds of nucleotide triphosphates to produce a modified single-stranded DNA target probe having a 3′ sticky end, hybridizing with a hairpin detection probe with complementary 3′ sticky ends, ligating to form a circular two-probe ligation product where the polynucleotide target sequence is not used as a template, degrading linear nucleic acid molecules after formation, amplifying the circular two-probe ligation product to produce a detectable amplicon, and detecting the presence of the circular two-probe ligation product by detecting the amplicon, thereby detecting the presence of the non-polynucleotide analyte.
Probe excess and repeating heating/cooling to increase modified target probe amount before circular ligation detection
Contacting a sample containing an unamplified polynucleotide analyte with a single-stranded DNA target probe having a hairpin structure under conditions where the amount of single-stranded DNA target probe is in substantial molar excess of the amount of the polynucleotide analyte, incubating in the presence of a polymerase and no more than three kinds of nucleotide triphosphates to produce a modified single-stranded DNA target probe having a 3′ sticky end, repeating steps (a) and (b) to increase the amount of modified single-stranded DNA target probe present in the sample including heating the sample after each step (b) to release modified single-stranded DNA target probe and cooling the sample as part of each step (a) to hybridize unreacted single-stranded DNA target probe with the target sequence, then hybridizing the modified target probe with a hairpin detection probe with complementary 3′ sticky ends and ligating to form a circular two-probe ligation product where the ligation reaction does not use the polynucleotide analyte as a template.
Across the independent claims, the coverage centers on creating a polymerase-extended, hairpin-structured 3′ sticky end on a target probe using no more than three kinds of nucleotide triphosphates, hybridizing with a complementary hairpin detection probe, and forming a circular two-probe ligation product without template use of the polynucleotide analyte. Claim coverage further includes post-ligation degradation and amplification to a detectable amplicon with amplicon-based detection, with additional independent coverage for detecting a non-polynucleotide analyte via an attached polynucleotide target sequence and for increasing modified target probe amount via substantial molar excess and repeating heating/cooling.
Stated Advantages
The polynucleotide analyte is not used as a template to form the circular two-probe ligation product.
Linear nucleic acid molecules are degraded after formation of the circular two-probe ligation product.
The circular two-probe ligation product is amplified to produce a detectable amplicon for detection.
A method is provided for detecting a non-polynucleotide analyte by attaching a polynucleotide target sequence to the non-polynucleotide analyte.
Modified single-stranded DNA target probe amount is increased by repeating steps including heating to release modified probe and cooling to hybridize unreacted probe.
Documented Applications
Detection of a target sequence in an unamplified polynucleotide analyte in a sample, including detection workflows that include circular two-probe ligation product formation and amplicon detection.
Detection of a non-polynucleotide analyte by attaching a polynucleotide target sequence to the non-polynucleotide analyte and performing the hairpin-based target probe and detection probe workflow.
Multiplex detection by mixing sample with hairpin-structured single-stranded DNA target probes for plurality of target sequences, adding single-stranded DNA detection probes, dividing into separate aliquots, and detecting the resulting circular two-probe ligation products.
RNA analyte detection using reverse transcriptase polymerase and promoter-dependent transcription with promoter elements and molecular beacon readouts.
miRNA multiplex detection.
SNP detection and multiplex SNP detection.
Protein-binding assay using antibody-linked polynucleotide markers.
solid support/array hybridization.
RNA single-tube/isothermal assays.
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