Polymerase extension at 3' terminus of PNA-DNA chimera
First Claim
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1. A labeled polymerase extended PNA-DNA chimera comprising the structure:
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Px-L-Ny 3′Extension wherein;
P is independently a PNA monomer;
X is an integer from 5 to 15;
L represents a covalent linkage between P and N;
each N is independently a nucleotide;
y is an integer from 1-15; and
3′Extension is the polymerase extension product comprising at least one non-radioisotopically labeled nucleotide wherein the label is 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.
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Abstract
The invention provides methods and kits 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.
16 Citations
6 Claims
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1. A labeled polymerase extended PNA-DNA chimera comprising the structure:
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Px-L-Ny 3′Extension
wherein;
P is independently a PNA monomer;
X is an integer from 5 to 15;
L represents a covalent linkage between P and N;
each N is independently a nucleotide;
y is an integer from 1-15; and
3′ Extension is the polymerase extension product comprising at least one non-radioisotopically labeled nucleotide wherein the label is 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.
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Current AssigneeCaifu Chen, Michael Egholm
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Original AssigneeCaifu Chen, Michael Egholm
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InventorsChen, Caifu, Egholm, Michael
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Application NumberUS10/618,077Publication NumberTime in Patent OfficeDaysField of SearchUS Class Current435/6CPC Class CodesC12Q 1/6853 using modified primers or t...C12Q 1/6858 Allele-specific amplificationC12Q 1/6869 Methods for sequencingC12Q 2525/107 incorporating a peptide nuc...C12Q 2537/143 Multiplexing, i.e. use of m...
