METHOD AND TEST SYSTEM FOR DETECTING THE C.1934dupG MUTATION IN THE ASXL1 GENE IN HEMOBLASTOSIS Russian patent published in 2022 - IPC C12Q1/6806 

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology. A test system for performing analyses of the c.1934dupG mutation in the ASXL1 gene is described, which consists of a polycarbonate tablet for PCR, where the wells contain a mixture for reaction under a layer of paraffin, where the control wells contain negative and positive samples for internal control of the study. At the same time, the reaction mixture consists of deionized DNA-free water, buffer solution, nucleotides, magnesium chloride, DNA polymerase, primers for annealing the patient’s DNA matrix in each well of the tablet. In addition, a method for performing analyses of the c.1934dupG mutation in the ASXL1 gene using the specified test system is described. Venous peripheral blood is taken into vacuum tubes from the patient. The procedure of isolation of genetic material is carried out using kits for isolation of DNA from peripheral blood. The genetic material (the patient’s DNA) is placed in a test system. The tablet is covered with an optically transparent film for sealing PCR tablets and the tablet is installed in a 96-well amplifier for real-time PCR. An amplification program is installed in the amplifier and a FAM channel is selected for fluorescence detection. At the end of the program, the efficiency of amplification of the normal and mutant matrix in each of the patients is analyzed by calculating the amplification efficiency index, comparing it with the average value of the amplification efficiency for the controls obtained in the experiment, which involves setting up laboratory equipment using control lines of the test system. At the same time, the biomaterial (the DNA under study) is not additionally purified after isolation from venous blood and is injected in a smaller volume (5 ng/mcl). The invention also improves the accuracy of the study of the c.1934dupG mutation in the ASXL1 gene by using two control lines to compare the collected DNA samples, in the control wells there is additionally control DNA and DNA with a 3% mutant matrix, respectively, marking the control wells for the ASXL1 study. In addition, venous blood is taken, which makes the process easier for medical personnel and less painful for the patient.

EFFECT: invention makes it possible to speed up the process of determining a mutation, since it requires fewer stages of research.

9 cl, 1 dwg, 2 tbl