Use of RNA/DNA chimeric primers for improved nucleic acid amplification reactions
First Claim
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1. A method of reducing or eliminating formation of artifacts and non-specific amplification products in a DNA-dependent DNA polymerase amplification reaction, the method comprising:
- conducting a nucleic acid amplification reaction using at least one RNA/DNA chimeric oligonucleotide as a forward or as a reverse primer or as a probe, and using a DNA-dependent DNA polymerase;
wherein the chimeric oligonucleotide comprises at least one ribonucleotide located within 10 nucleotides adjacent to the 3′
end of the chimeric oligonucleotide;
wherein the at least one ribonucleotide impedes DNA synthesis from primer-primer dimers or primer-probe dimers; and
wherein no two ribonucleotides in the chimeric oligonucleotide are adjacent to one another.
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Abstract
Methods are provided for amplification of a nucleic acid sequence. The method use RNA/DNA chimeric oligonucleotides as primers. The primers have RNA residues scattered along their length and no two ribonucleotides in the prime are adjacent to one another. The methods are useful for reducing non-specific amplification products, such as primer dimers. The invention also provides kits comprising RNA/DNA chimeric oligonucleotide primers for practicing the amplification methods.
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Citations
19 Claims
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1. A method of reducing or eliminating formation of artifacts and non-specific amplification products in a DNA-dependent DNA polymerase amplification reaction, the method comprising:
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conducting a nucleic acid amplification reaction using at least one RNA/DNA chimeric oligonucleotide as a forward or as a reverse primer or as a probe, and using a DNA-dependent DNA polymerase; wherein the chimeric oligonucleotide comprises at least one ribonucleotide located within 10 nucleotides adjacent to the 3′
end of the chimeric oligonucleotide;wherein the at least one ribonucleotide impedes DNA synthesis from primer-primer dimers or primer-probe dimers; and wherein no two ribonucleotides in the chimeric oligonucleotide are adjacent to one another. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A kit for carrying out a DNA-dependent DNA polymerase amplification reaction comprising:
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(i) at least one RNA/DNA chimeric oligonucleotide as a forward or as a reverse primer or as a probe; wherein the chimeric oligonucleotide comprises at least one ribonucleotide located within 10 nucleotides adjacent to the 3′
end of the chimeric oligonucleotide;wherein the at least one ribonucleotide impedes DNA synthesis from primer-primer dimers or primer-probe dimers; and wherein no two ribonucleotides in the chimeric oligonucleotide are adjacent to one another; (ii) a DNA-dependent DNA polymerase; (iii) the necessary reagents and buffers to carry out the amplification reaction. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18, 19)
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Specification