Primer extension using modified nucleotides
First Claim
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1. A method for inhibiting template independent primer extension, comprising:
- employing a primer extension reaction using i) a polymerase, ii) a template, iii) a plurality of primers having a palindromic nucleotide sequence, by;
hybridizing a first primer of the plurality of primers to the template so as to form a primer-template duplex;
hybridizing a second of the plurality of primers to a third of the plurality of primers so as to form a primer-primer duplex, and each of the plurality of primers comprise one or more modified nucleotides at or near the 3′
or 5′
end of the palindromic nucleotide sequence that inhibit(s) template independent primer extension of the primer-primer duplex, wherein the polymerase extends the primer of the primer-template duplex and the one or more modified nucleotides of the plurality of primers is modified at the 2′
position and the first, second, and third of the plurality of primers have the same nucleotide sequence.
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Abstract
The present invention provides methods and compositions that reduce target-independent primer extension or enhance template dependent primer extension. The methods and compositions of the present invention are applicable not only in PCR, but also in nucleic acid sequencing, genotyping, and other applications employing extension of a primer in a target-dependent manner.
35 Citations
16 Claims
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1. A method for inhibiting template independent primer extension, comprising:
- employing a primer extension reaction using i) a polymerase, ii) a template, iii) a plurality of primers having a palindromic nucleotide sequence, by;
hybridizing a first primer of the plurality of primers to the template so as to form a primer-template duplex; hybridizing a second of the plurality of primers to a third of the plurality of primers so as to form a primer-primer duplex, and each of the plurality of primers comprise one or more modified nucleotides at or near the 3′
or 5′
end of the palindromic nucleotide sequence that inhibit(s) template independent primer extension of the primer-primer duplex, wherein the polymerase extends the primer of the primer-template duplex and the one or more modified nucleotides of the plurality of primers is modified at the 2′
position and the first, second, and third of the plurality of primers have the same nucleotide sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
- employing a primer extension reaction using i) a polymerase, ii) a template, iii) a plurality of primers having a palindromic nucleotide sequence, by;
Specification
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Current AssigneeOrchid Cellmark Incorporated (Laboratory Corporation Of America Holdings)
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Original AssigneeOrchid Cellmark Incorporated (Laboratory Corporation Of America Holdings)
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InventorsGelfand, Craig A., Swenson, Rolf E., Zhao, Xiaodong
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Primary Examiner(s)Horlick; Kenneth R.
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Assistant Examiner(s)Tung; Joyce
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Application NumberUS10/119,520Publication NumberTime in Patent Office1,848 DaysField of Search435/6, 435/91.2, 435/395, 536/24.33, 536/23.1, 536/25.32, 536/23.2US Class Current435/6.11CPC Class CodesC12Q 1/6848 characterised by the means ...C12Q 1/686 Polymerase chain reaction [...C12Q 1/6869 Methods for sequencingC12Q 2525/117 incorporating modified baseC12Q 2533/101 Primer extensionC12Q 2600/156 Polymorphic or mutational m...
