ALLELE-SPECIFIC AMPLIFICATION USING COMPOSITION OF OVERLAPPING OLIGONUCLEOTIDES AS NON-ALLELE-SPECIFIC PRIMER AND ALLELE-SPECIFIC BLOCKER

摘要

FIELD: biotechnologies.;SUBSTANCE: invention relates to the biotechnology. Described is a composition of oligonucleotides for amplification of a target sequence. Composition contains an oligonucleotide as a blocker, including a functional group or an uncomplementary section of sequence at 3'-end or nearby it, which prevents enzymatic elongation. Oligonucleotide as a blocker includes a first sequence comprising a neutral to the target sub-sequence and a blocking variable subsequence, where the blocking variable subsequence is flanked at its 3'- and 5'-ends of a subsequence which is neutral with respect to the target and is inextricable with a neutral to the target of the sub-sequence. Oligonucleotide as the first primer is sufficient to induce enzymatic elongation. Oligonucleotide as a first primer includes a second sequence, where the second sequence is overlapped with a neutral to the target subsequence of the oligonucleotide as a blocker for at least 5 nucleotides, so that the second sequence includes an overlapping sub-sequence and a nonoverlapping sub-sequence, and the second sequence does not include a blocking variable subsequence, second sequence creates standard free hybridisation energy (ΔG^ο_PT), and the first sequence generates standard free hybridisation energy (ΔG^ο_BT), which satisfies the following condition: +2 kcal/mol ≥ΔG^ο_PT-ΔG^ο_BT≥-8 kcal/mol. Concentration of the oligonucleotide as a blocker is approximately 2 - approximately 10,000 times higher than the concentration of the oligonucleotide as the first primer.;EFFECT: invention extends range of means for amplification.;47 cl, 14 dwg, 1 tbl, 11 ex