Methods and compositions for the manufacture of molecular beacons
First Claim
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1. A method of making one or more molecular beacons or molecular beacon components, comprising:
- providing a first oligonucleotide or peptide nucleic acid (PNA) corresponding to a first subsequence of a molecular beacon;
providing a second oligonucleotide or PNA corresponding to a second subsequence of the molecular beacon;
providing a third oligonucleotide or PNA corresponding to a third subsequence of the molecular beacon; and
, ligating the first, second and third oligonucleotides or PNAs together, thereby forming the molecular beacon or the molecular beacon component wherein the first, second and third oligonucleotides are ligated using two template oligonucleotides to align the first, second and third oligonucleotides for ligation.
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Abstract
Ligation-based assembly of oligonucleotides to produce molecular beacons is provided. Formation of molecular beacons is monitored to improve reaction yield and efficiency and to permit optimization of structurally similar molecular beacons. Ligation mixtures and libraries of molecular beacon components are also provided. Methods of detecting juxtaposed nucleic acids using modular molecular beacons such as intron-exon junctions are provided.
16 Citations
39 Claims
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1. A method of making one or more molecular beacons or molecular beacon components, comprising:
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providing a first oligonucleotide or peptide nucleic acid (PNA) corresponding to a first subsequence of a molecular beacon;
providing a second oligonucleotide or PNA corresponding to a second subsequence of the molecular beacon;
providing a third oligonucleotide or PNA corresponding to a third subsequence of the molecular beacon; and
,ligating the first, second and third oligonucleotides or PNAs together, thereby forming the molecular beacon or the molecular beacon component wherein the first, second and third oligonucleotides are ligated using two template oligonucleotides to align the first, second and third oligonucleotides for ligation. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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20. A device comprising:
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a reaction region comprising a ligation mixture therein, the ligation mixture comprising a first oligonucleotide or PNA comprising a label moiety;
a second oligonucleotide or PNA comprising a quenching moiety that quenches the label moiety when placed proximal to or in contact with the label moiety; and
a third oligonucleotide or PNA;
wherein ligation of the first, second and third oligonucleotides or PNAs results in formation of a molecular beacon and wherein the ligation mixture further comprises fourth and fifth oligonucleotides which provide templates for ligation of the first, second and third oligonucleotides;
a ligase; and
,a detector proximal to the reaction region, which detector detects one or more ligation-dependent change in an output of the label moiety. - View Dependent Claims (21, 22, 23, 24, 25, 26, 27, 28, 29, 30)
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31. A library of molecular beacon components, comprising:
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a set of a plurality of hairpin loop oligonucleotides or PNAs, each of the plurality of hairpin loop oligonucleotides or PNAs comprising a subsequence of at least one molecular beacon, the subsequence comprising less than all of the molecular beacon;
at least one first label and at least one first label quenching oligonucleotide or PNA, which first label and first label quenching oligonucleotides or PNAs each comprise at least one label or label quenching moiety and at least two template oligonucleotides, which at least two template oligonucleotides collectively hybridize to a first of the hairpin loop oligonucleotides, the first or the label quenching oligonucleotides and the first of the label oligonucleotides, wherein ligation of the first hairpin loop oligonucleotide, first label quenching oligonucleotide and first label oligonucleotide, when hybridized to the two template oligonucleotides, produces a molecular beacon. - View Dependent Claims (32, 33, 34, 35, 36)
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37. A method of detecting a juxtaposition of two or more target subsequences in a target nucleic acid, comprising:
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forming a molecular beacon by ligating first, second and third oligonucleotides together while the first, second and third oligonucleotides are hybridized to first second template oligonucleotides, thereby forming a molecular beacon, wherein the first, second and third oligonucleotides collectively comprise regions of complementarity to first and second target subsequences;
hybridizing the resulting molecular beacon to the target nucleic add; and
,detecting target-specific hybridization of the molecular beacon to the first and second subsequences. - View Dependent Claims (38, 39)
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Specification