Methods for preparing tagging oligonucleotides
First Claim
1. A method for preparing a tagging oligonucleotide comprising a targeting portion comprising a hybridizable-complementary nucleotide sequence to a target nucleic acid sequence and a tagging portion comprising a non-hybridizable-non-complementary nucleotide sequence to the target nucleic acid sequence, comprising:
- (a) selecting the hybridizable-complementary nucleotide sequence to the target nucleic acid sequence for the targeting portion and the non-hybridizable-non-complementary nucleotide sequence to the target nucleic acid sequence for the tagging portion;
wherein the tagging portion comprises a first tagging part of 3-8 nucleotides in length adjacent to the targeting portion and a second tagging part of 4-40 nucleotides in length adjacent to the first tagging part;
the non-hybridizable-non-complementary nucleotide sequence for the tagging portion is selected not to be hybridized with the target nucleic acid sequence;
wherein a non-hybridizable-non-complementary nucleotide sequence of the first tagging part is selected by an independent non-complementarity level such that a sequence with a non-complementarity level satisfying a predetermined threshold value criterion is selected as the non-hybridizable-non-complementary nucleotide sequence of the first tagging part; and
(b) preparing the tagging oligonucleotide comprising (i) the targeting portion comprising the selected hybridizable-complementary nucleotide sequence and (ii) the tagging portion comprising the selected non-hybridizable-non-complementary nucleotide sequence.
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Abstract
The present invention relates to technologies for preparing a tagging oligonucleotide. By analyzing exquisitely a non-complementarity level of a first tagging part, the first aspect of the present invention permits to more efficiently and easily select a suitable tagging sequence among a multitude of sequences generated theoretically. In addition, according to the second aspect of the present invention, when a nucleotide sequence for a tagging portion is first selected, one or more regions in a target nucleic acid sequence having a non-complementarity level to the nucleotide sequence for the tagging portion are found, and then a nucleotide sequence for a targeting portion is determined, tagging oligonucleotides for detecting various target nucleic acid sequences can prepared by using the fewest number of nucleotide sequences for the tagging portion and a third template.
10 Citations
18 Claims
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1. A method for preparing a tagging oligonucleotide comprising a targeting portion comprising a hybridizable-complementary nucleotide sequence to a target nucleic acid sequence and a tagging portion comprising a non-hybridizable-non-complementary nucleotide sequence to the target nucleic acid sequence, comprising:
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(a) selecting the hybridizable-complementary nucleotide sequence to the target nucleic acid sequence for the targeting portion and the non-hybridizable-non-complementary nucleotide sequence to the target nucleic acid sequence for the tagging portion;
wherein the tagging portion comprises a first tagging part of 3-8 nucleotides in length adjacent to the targeting portion and a second tagging part of 4-40 nucleotides in length adjacent to the first tagging part;
the non-hybridizable-non-complementary nucleotide sequence for the tagging portion is selected not to be hybridized with the target nucleic acid sequence;
wherein a non-hybridizable-non-complementary nucleotide sequence of the first tagging part is selected by an independent non-complementarity level such that a sequence with a non-complementarity level satisfying a predetermined threshold value criterion is selected as the non-hybridizable-non-complementary nucleotide sequence of the first tagging part; and(b) preparing the tagging oligonucleotide comprising (i) the targeting portion comprising the selected hybridizable-complementary nucleotide sequence and (ii) the tagging portion comprising the selected non-hybridizable-non-complementary nucleotide sequence. - View Dependent Claims (3, 6, 7, 10, 12, 13, 14, 15, 16)
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2. A method for preparing a tagging oligonucleotide comprising a targeting portion comprising a hybridizable-complementary nucleotide sequence to a target nucleic acid sequence and a tagging portion comprising a non-hybridizable-non-complementary nucleotide sequence to the target nucleic acid sequence, comprising:
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(a) providing a nucleotide sequence for the tagging portion;
wherein the tagging portion comprises a first tagging part adjacent to the targeting portion and a second tagging part adjacent to the first tagging part;(b) selecting in the target nucleic acid sequence one or more non-hybridizable-non-complementary regions to the nucleotide sequence for the tagging portion by (i) selecting at least one region in the target nucleic acid sequence and (ii) evaluating a non-complementarity level of the selected at least one region to the nucleotide sequence for the tagging portion; (c) selecting a nucleotide sequence for the targeting portion by (i) selecting a nucleotide sequence with a predetermined length in the target nucleic acid sequence that is adjacent to the non-hybridizable-non-complementary region to the nucleotide sequence of the tagging portion and (ii) selecting a hybridizable-complementary nucleotide sequence to the selected nucleotide sequence in the target nucleic acid as the nucleotide sequence for the targeting portion; and (d) preparing the tagging oligonucleotide comprising the nucleotide sequence of the tagging portion provided in the step (a) and the nucleotide sequence of the targeting portion selected in the step (c). - View Dependent Claims (4, 5, 8, 9, 11)
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17. A computer readable storage medium containing instructions to configure a processor to perform a method for preparing a tagging oligonucleotide comprising a targeting portion comprising a hybridizable-complementary nucleotide sequence to a target nucleic acid sequence and a tagging portion comprising a non-hybridizable-non-complementary nucleotide sequence to the target nucleic acid sequence, the method comprising:
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(a) selecting the hybridizable-complementary nucleotide sequence to the target nucleic acid sequence for the targeting portion and the non-hybridizable-non-complementary nucleotide sequence to the target nucleic acid sequence for the tagging portion;
wherein the tagging portion comprises a first tagging part of 3-8 nucleotides in length adjacent to the targeting portion and a second tagging part of 4-40 nucleotides in length adjacent to the first tagging part;
the non-hybridizable-non-complementary nucleotide sequence for the tagging portion is selected not to be hybridized with the target nucleic acid sequence;
wherein a non-hybridizable-non-complementary nucleotide sequence of the first tagging part is selected by an independent non-complementarity level such that a sequence with a non-complementarity level satisfying a predetermined threshold value criterion is selected as the non-hybridizable-non-complementary nucleotide sequence of the first tagging part; and(b) preparing the tagging oligonucleotide comprising (i) the targeting portion comprising the selected hybridizable-complementary nucleotide sequence and (ii) the tagging portion comprising the selected non-hybridizable-non-complementary nucleotide sequence.
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18. A computer readable storage medium containing instructions to configure a processor to perform a method for preparing a tagging oligonucleotide comprising a targeting portion comprising a hybridizable-complementary nucleotide sequence to a target nucleic acid sequence and a tagging portion comprising a non-hybridizable-non-complementary nucleotide sequence to the target nucleic acid sequence, the method comprising:
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(a) providing a nucleotide sequence for the tagging portion;
wherein the tagging portion comprises a first tagging part adjacent to the targeting portion and a second tagging part adjacent to the first tagging part;(b) selecting in the target nucleic acid sequence one or more non-hybridizable-non-complementary regions to the nucleotide sequence for the tagging portion by (i) selecting at least one region in the target nucleic acid sequence and (ii) evaluating a non-complementarity level of the selected at least one region to the nucleotide sequence for the tagging portion; (c) selecting a nucleotide sequence for the targeting portion by (i) selecting a nucleotide sequence with a predetermined length in the target nucleic acid sequence that is adjacent to the non-hybridizable-non-complementary region to the nucleotide sequence of the tagging portion and (ii) selecting a hybridizable-complementary nucleotide sequence to the selected nucleotide sequence in the target nucleic acid as the nucleotide sequence for the targeting portion; and (d) preparing the tagging oligonucleotide comprising the nucleotide sequence of the tagging portion provided in the step (a) and the nucleotide sequence of the targeting portion selected in the step (c).
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Specification