Method for reducing artifacts in nucleic acid amplification
First Claim
1. A method of reducing formation of artifacts in a nucleic acid amplification reaction, the method comprisingconducting a nucleic acid amplification reaction using a template-deficient oligonucleotide as a primer and using a polymerase,wherein the template-deficient oligonucleotide comprises one or more template-deficient nucleotides,wherein the number and composition of template-capable nucleotides 3′
- of the template-deficient nucleotide closest to the 3′
end of the template-deficient oligonucleotide is sufficient to allow the template-capable nucleotides 3′
of the template-deficient nucleotide closest to the 3′
end alone to effectively prime nucleic acid synthesis in the nucleic acid amplification reaction reaction,wherein the template-deficient oligonucleotide is template-deficient when used in a nucleic acid amplification reaction with the polymerase.
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Abstract
Disclosed are compositions and methods useful for reducing the formation of artifacts during nucleic acid amplification reactions. The method uses special oligonucleotides, referred to herein as template-deficient oligonucleotides, that cannot serve as a template for nucleic acid synthesis over part of their length. This prevents the oligonucleotides from serving as effective templates in the formation of artifacts. The disclosed method involves using a template-deficient oligonucleotide as at least one of the oligonucleotides (preferably a primer) in a nucleic acid amplification reaction, where the template-deficient oligonucleotide comprises one or more template-deficient nucleotides, preferably at or near the 5′ end of the template-deficient oligonucleotide. The disclosed method is useful for reducing artifacts in any nucleic acid amplification reaction involving oligonucleotides. In a preferred form of the method the nucleic acid amplification reaction does not involve thermal cycling. The disclosed method is effective at reducing non-cycle oligonucleotide-based artifacts. Also disclosed are kits useful for reducing artifacts in nucleic acid amplification reactions. The disclosed kits include a template-deficient oligonucleotide, wherein the template-deficient oligonucleotide comprises one or more template-deficient nucleotides, and a nucleic acid polymerase.
36 Citations
25 Claims
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1. A method of reducing formation of artifacts in a nucleic acid amplification reaction, the method comprising
conducting a nucleic acid amplification reaction using a template-deficient oligonucleotide as a primer and using a polymerase, wherein the template-deficient oligonucleotide comprises one or more template-deficient nucleotides, wherein the number and composition of template-capable nucleotides 3′ - of the template-deficient nucleotide closest to the 3′
end of the template-deficient oligonucleotide is sufficient to allow the template-capable nucleotides 3′
of the template-deficient nucleotide closest to the 3′
end alone to effectively prime nucleic acid synthesis in the nucleic acid amplification reaction reaction,wherein the template-deficient oligonucleotide is template-deficient when used in a nucleic acid amplification reaction with the polymerase. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 19, 20, 21)
- of the template-deficient nucleotide closest to the 3′
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16. The method of 14 wherein the nucleic acid amplification reaction involves the polymerase chain reaction.
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18. The method of 17 wherein the nucleic acid amplification is rolling circle amplification.
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22. A method of reducing formation of artifacts in a nucleic acid amplification reaction, the method comprising
conducting a nucleic acid amplification reaction using a template-deficient oligonucleotide as a primer and using a polymerase, wherein the template-deficient oligonucleotide comprises one or more template-deficient nucleotides, wherein the one or more adjacent template-deficient nucleotides are within three nucleotides of the 5′ - end of the template-deficient oligonucleotide,
wherein the number and composition of template-capable nucleotides 3′
of the template-deficient nucleotide closest to the 3′
end of the template-deficient oligonucleotide is sufficient to allow the template-capable nucleotides 3′
of the template-deficient nucleotide closest to the 3′
end alone to effectively prime nucleic acid synthesis in the nucleic acid amplification reaction reaction,wherein the template-deficient oligonucleotide is template-deficient when used in a nucleic acid amplification reaction with the polymerase. - View Dependent Claims (23)
- end of the template-deficient oligonucleotide,
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24. A method of reducing formation of artifacts in a nucleic acid amplification reaction, the method comprising
conducting a nucleic acid amplification reaction using a template-deficient oligonucleotide as a primer and using a polymerase, wherein the template-deficient oligonucleotide comprises one or more template-deficient nucleotides, wherein the modified nucleotides are abasic nucleotides, wherein the number and composition of template-capable nucleotides 3′ - of the template-deficient nucleotide closest to the 3′
end of the template-deficient oligonucleotide is sufficient to allow the template-capable nucleotides 3′
of the template-deficient nucleotide closest to the 3′
end alone to effectively prime nucleic acid synthesis in the nucleic acid amplification reaction,wherein the template-deficient oligonucleotide is template-deficient when used in a nucleic acid amplification reaction with the polymerase.
- of the template-deficient nucleotide closest to the 3′
-
25. A method of reducing formation of artifacts in a nucleic acid amplification reaction, the method comprising
conducting a nucleic acid amplification reaction using a template-deficient oligonucleotide as a primer and using a polymerase, wherein the template-deficient oligonucleotide comprises one or more template-deficient nucleotides, wherein the one or more adjacent template-deficient nucleotides are within three nucleotides of the 5′ - end of the template-deficient oligonucleotide, wherein the modified nucleotides are abasic nucleotides,
wherein the number and composition of template-capable nucleotides 3′
of the template-deficient nucleotide closest to the 3′
end of the template-deficient oligonucleotide is sufficient to allow the template-capable nucleotides 3′
of the template-deficient nucleotide closest to the 3′
end alone to effectively prime nucleic acid synthesis in the nucleic acid amplification reaction,wherein the template-deficient oligonucleotide is template-deficient when used in a nucleic acid amplification reaction with the polymerase.
- end of the template-deficient oligonucleotide, wherein the modified nucleotides are abasic nucleotides,
Specification