Terminus-Specific DNA Modification Using Random-Sequence Template Oligonucleotides
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Abstract
A method is provided for analyzing DNA molecules having unknown 3′ terminal sequences. The method involves contacting a DNA molecule with a plurality of template oligonucleotides blocked at their 3′ termini such that the template oligonucleotides are not extendable by DNA polymerase. The 3′ proximal portions of each of the template oligonucleotides comprise a region of random sequence and the 5′ proximal portions of each of the template oligonucleotides comprise the complement of a tag sequence. The DNA molecule and the template oligonucleotides are combined under conditions wherein the 3′ terminus of the DNA molecule hybridizes to the 3′ proximal portion of a template oligonucleotide and is extended by a DNA polymerase to produce a DNA molecule comprising a 3′ terminal tag sequence, and wherein the template oligonucleotide is not extended.
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Citations
29 Claims
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1-20. -20. (canceled)
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21. A method of analyzing a DNA molecule having an unknown 3′
- terminal sequence, comprising;
a) providing a sample comprising at least one DNA molecule having a 3′
terminus of unknown sequence;b) combining said sample with a mixture comprising a plurality of template oligonucleotides blocked at their 3′
termini such that said plurality of template oligonucleotides are not extendable by a DNA- or RNA-directed DNA polymerase, wherein 3′
proximal portions of said plurality of template oligonucleotides each comprise a region of random sequence and wherein 5′
proximal portions of said plurality of template oligonucleotides each comprise the complement of a tag sequence, and under conditions wherein;i) said 3′
terminus of said DNA molecule hybridizes to the 3′
proximal portion of a template oligonucleotide, andii) said 3′
terminus of said DNA molecule is extended by a DNA- or RNA-directed DNA polymerase to produce a DNA molecule comprising a 3′
terminal tag sequence, andiii) said template oligonucleotide is not extended by said DNA- or RNA-directed DNA polymerase; and c) detecting said DNA comprising said 3′
terminal tag sequence. - View Dependent Claims (22, 23, 24, 25, 26, 27, 28, 29)
- terminal sequence, comprising;
Specification