METHOD FOR PRODUCTION OF MARKER LADDERS FOR GEL ELECTROPHORETIC DETERMINATION OF NUCLEIC ACID FRAGMENT SIZES Russian patent published in 2017 - IPC C12Q1/68 

FIELD: biotechnology.

SUBSTANCE: method for producing a special type of consumables for biomedical research, length markers of nucleic acid fragments (DNA and RNA marker ladders). The invention consists in testing of DNA and RNA fragments, constituting the ladder via amplification reaction by a "ring rolling" in rolling circle amplification embodiment. As amplification matrix, a single-stranded DNA ring molecule of desired size L is used (where L is the number of nucleotides), which is formed from a synthetic oligonucleotide of size L by intramolecular ligation. Ring ligation product is used as a matrix for annealing and elongation of "seed" primer 1, and primer 2 present in the reaction mixture provides for amplification starting according to amplification type. For polymerization reaction of polynucleotide chain, DNA or RNA polymerases are used having chain displacing activity, amplification is carried out in an isothermal or a quasi-isothermal mode. When adding to the reaction mixture of small amounts of restrictases and nickases, growing chains splitting may be achieved according to recognition sites for these enzymes, thereby reducing the amount of very extended amplification products in favour of shorter. To produce RNA marker ladders, used deoxyribo- ribonucleoside triphosphates and the mixture of DNA and RNA polymerases are used. To produce marker ladders of fragment sizes not multiple of the size of the ring matrix, primers with 5'-terminal sequence of any desired length and non-complementary annular original matrix are used. Another way to produce marker ladders woth the size of fragments not multiple of the size of the ring matrix is a simple mixture of products obtained by amplification of ring matrices of different sizes. The proposed method allows to obtain marker ladders in a significant amount with any size increments. They can be used in medical diagnostic laboratories, testing laboratories and research and development laboratories during implementation of all types of all gel electrophoretic analysis of nucleic acids.

EFFECT: use of marker ladders produced in the manner described above, is identical to that for all commercially available marker ladders.

5 cl, 5 dwg, 9 ex