Polymerase extension at 3′ terminus of PNA-DNA chimera
First Claim
1. A method of producing a non-radioisotopically labelled chimeric extension product comprising the step of enzymatically extending a PNA-DNA chimera in the presence of a template nucleic acid, a polymerase and a primer extension reagent, wherein said primer extension reagent comprises a nucleotide 5′
- -triphosphate capable of effecting enzymatic chimera primer extension, the PNA-DNA chimera or the nucleotide 5′
-triphosphate is labelled with a non-radioisotopic label selected from the group consisting of a fluorescent dye, a fluorescence quencher, a hybridization stabilizer, an energy-transfer dye pair, an electrophoretic mobility modifier, a chemiluminescent dye, an amino acid, a protein, a peptide, an enzyme, and an affinity ligand, andthe PNA-DNA chimera has the structure;
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Abstract
The invention provides methods and a kit for primer extension of PNA-DNA chimera from template nucleic acids using polymerases, nucleotide 5′-triphosphates, and primer extension reagents. Structural requirements of the chimera for primer extension include 5 to 15 contiguous PNA monomer units, 3 or more contiguous nucleotides, and a 3′ hydroxyl terminus. The chimera and/or a nucleotide is labelled with fluorescent dyes or other labels. The methods include DNA sequencing, DNA fragment analysis, reverse transcription, mini-sequencing, chromosome labelling, amplification, and single nucleotide polymorphism (SNP) detection.
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Citations
43 Claims
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1. A method of producing a non-radioisotopically labelled chimeric extension product comprising the step of enzymatically extending a PNA-DNA chimera in the presence of a template nucleic acid, a polymerase and a primer extension reagent, wherein said primer extension reagent comprises a nucleotide 5′
- -triphosphate capable of effecting enzymatic chimera primer extension, the PNA-DNA chimera or the nucleotide 5′
-triphosphate is labelled with a non-radioisotopic label selected from the group consisting of a fluorescent dye, a fluorescence quencher, a hybridization stabilizer, an energy-transfer dye pair, an electrophoretic mobility modifier, a chemiluminescent dye, an amino acid, a protein, a peptide, an enzyme, and an affinity ligand, andthe PNA-DNA chimera has the structure;
- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33)
- -triphosphate capable of effecting enzymatic chimera primer extension, the PNA-DNA chimera or the nucleotide 5′
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34. A kit for primer extension comprising:
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a PNA-DNA chimera primer, said primer comprising 5 to 15 contiguous PNA monomer units, 3 to 15 contiguous nucleotides, and a 3′
hydroxyl terminus;
one or more nucleotide 5′
-triphosphates and;
a polymerase enzyme,wherein the chimera primer or a nucleotide 5′
-triphosphate is non-radioisotopically labelled.- View Dependent Claims (35)
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36. A method of sequencing a template nucleic acid, comprising the steps of:
a) generating a labelled primer extension product by enzymatically extending a primer-template nucleic acid hybrid in the presence of a polymerase and a terminating nucleotide 5′
-triphosphate, wherein said primer is a PNA-DNA chimera and either said primer or said terminating nucleotide 5′
-triphosphate is detectably and non-radioisotopically labelled with a non-radioisotopic label selected from the group consisting of a fluorescent dye, a fluorescence quencher, a hybridization stabilizer, an energy-transfer dye pair, an electrophoretic mobility modifier, a chemiluminescent dye, an amino acid, a protein, a peptide, an enzyme, and an affinity ligand, and the PNA-DNA chimera has the structure;
- View Dependent Claims (37, 38, 39, 40)
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41. A method of reverse transcription comprising the step of generating labelled primer extension products by enzymatically extending a primer-template RNA hybrid in the presence of a reverse transcriptase, a mixture of enzymatically-extendable nucleotide 5′
- -triphosphates capable of supporting continuous primer extension, wherein said primer is a PNA-DNA chimera and either said primer or a nucleotide 5′
-triphosphate is non-radioisotopically labelled with a non-radioisotopic label selected from the group consisting of a fluorescent dye, a fluorescence quencher, a hybridization stabilizer, an energy-transfer dye pair, an electrophoretic mobility modifier, a chemiluminescent dye, an amino acid, a protein, a peptide, an enzyme, and an affinity ligand, andthe PNA-DNA chimera has the structure;
- -triphosphates capable of supporting continuous primer extension, wherein said primer is a PNA-DNA chimera and either said primer or a nucleotide 5′
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42. A method of DNA amplification comprising the steps of:
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a) generating labelled amplification products by enzymatically extending a primer-template nucleic acid hybrid in the presence of two primers each of which is capable of hybridizing to the template and wherein one or both of which is a PNA-DNA chimera primer, a DNA polymerase and a mixture of enzymatically-extendable nucleotide 5′
-triphosphates capable of supporting continuous primer extension, wherein either said primers or nucleotide 5′
-triphosphates are non-radioisotopically labelled with a non-radioisotopic label selected from the group consisting of a fluorescent dye, a fluorescence quencher, a hybridization stabilizer, an energy-transfer dye pair, an electrophoretic mobility modifier, a chemiluminescent dye, an amino acid, a protein, a peptide, an enzyme, and an affinity ligand, andthe PNA-DNA chimera has the structure;
- View Dependent Claims (43)
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Specification