Simple, effective and fast way of enzymatic modification and synthesis of nucleic acids IN VITRO USING MICROWAVE

摘要

1. a simple, effective and fast way to u0444u0435u0440u043cu0435u043du0442u0430u0442u0438u0432u043du043eu0439 modification and synthesis of nucleic acid in vitro using continuous and a microwave u0438u0437u043b u0443u0447u0435u043du0438u00a0, where the method includes u0441u0435u0431u00a0 stage.;(a) u043fu043eu043bu0443u0447u0435u043du0438u00a0 reaction mixture containing (i) an enzyme selected from the group u0441u043eu0441u0442u043eu00a0u0449u0435u0439 of u0440u0435u0441u0442u0440u0438u043au0446u0438u043eu043du043du043eu0439 endonuclease, u043bu0438u0433u0430u0437u044b, kinase, phosphatase, reverse u0442u0440u0430u043du0441u043au0440u0438u043f bowls of adp ribose polymerase, dna and rna adp ribose polymerase, (ii) u0431u0438u043eu043cu043eu043bu0435u043au0443u043bu0443 selected from dna, rna or u043eu043bu0438u0433u043eu043du0443u043au043bu0435u043eu0442u0438u0434u043eu0432, as the enzyme substrate u0434u043bu00a0, (iii) buffer, u043fu043eu0434u0445u043eu0434u00a0u0449 yi u0434u043bu00a0 specific enzyme, and (iv) other ingredientsin a small tube u044du043fu043fu0435u043du0434u043eu0440u0444u0430 or on a small piece of u043fu0430u0440u0430u0444u0438u043bu044cu043cu0430,;(b) placement of the tube u044du043fu043fu0435u043du0434u043eu0440u0444u0430 or piece of u043fu0430u0440u0430u0444u0438u043bu044cu043cu0430 with reaction mixture in the microwave oven.;(c) the continuous exposure of the tube u044du043fu043fu0435u043du0434u043eu0440u0444u0430 or piece of u043fu0430u0440u0430u0444u0438u043bu044cu043cu0430 microwaves at a frequency of from 2300 to 2500 mhz and power output from 600 to 900 w,;(d) during the period from 5 to 120 c;(e) stops the reaction, adding u0442u0435u0442u0440u0430u0430u0446u0435u0442u0430u0442u0430 u044du0442u0438u043bu0435u043du0434u0438u0430u043cu0438u043du0430 (EDTA) and / or heating at u0432u043eu0434u00a0u043du043eu0439 bath with rough crystallized structure within 3 - 15 min with the addition of u0433u0435u043bu00a0 bearing dye;(d) analysis of the reaction product (s) by electrophoresis on u0430u0433u0430u0440u043eu0437u043du043eu043c gehle, u0430u0432u0442u043eu0440u0430u0434u0438u043eu0433u0440u0430u0444u0438u0438, counting radioactive tags and other traditional methods.;2. method for 1, where u043du0443u043au043bu0435u0438u043du043eu0432u0430u00a0 acid selected from dna, rna and u043eu043bu0438u0433u043eu043du0443u043au043bu0435u043eu0442u0438u0434u0430.;3. method for 1, where other ingredients selected from the group u0441u043eu0441u0442u043eu00a0u0449u0435u0439 of MgCl2, MgSO4, control valve, KCl, CH3COOK, u0442u0435u0442u0440u0430u0430u0446u0435u0442u0430u0442u0430 u044du0442u0438u043bu0435u043du0434u0438u0430u043cu0438u043du0430, u0434u0438u0442u0438u043eu0442u0440u0435u0438u0442u043eu043bu0430, u0441u043fu0435u0440u043cu0438u0434u0438u043du0430, bsa, triton x - 100, nook u043bu0435u043eu0442u0438u0434u043eu0432, matrix matrix, dna, rna and u043eu043bu0438u0433u043eu043du0443u043au043bu0435u043eu0442u0438u0434u043du044bu0445 u043fu0440u0430u0439u043cu0435u0440u043eu0432.;4. method for 1, where u0440u0435u0441u0442u0440u0438u043au0446u0438u043eu043du043du0430u00a0 u044du043du0434u043eu043du0443u043au043bu0435u0430u0437u0430 selected from the group u0441u043eu0441u0442u043eu00a0u0449u0435u0439 of Hind iii, BamHI, EcoRI, hae iii, Bgl ii, Pst i, Bst e ii and Bst ni.;5. method for 1, where as the u043bu0438u0433u0430u0437u044b using dna u043bu0438u0433u0430u0437u0443 t4.;6. method for 1, where as the kinase using u043fu043eu043bu0438u043du0443u043au043bu0435u043eu0442u0438u0434u043au0438u043du0430u0437u0443 t4 (TPNK).;7. way to 1, where the use of calf intestinal phosphatase respond u0444u043eu0441u0444u0430u0442u0430u0437u0443 (cip).;8. method for 1, where u043fu043eu043bu0438u043cu0435u0440u0430u0437u0430 selected from the group u0441u043eu0441u0442u043eu00a0u0449u0435u0439 dna adp ribose polymerase i from e. coli, dna fragment u043au043bu0435u043du043eu0432u0430 adp ribose polymerase i e. coli, adp ribose polymerase t7 rna and rna SP6 adp ribose polymerase.;9. method for 1, where as the inverse u0442u0440u0430u043du0441u043au0440u0438u043fu0442u0430u0437u044b use the u0442u0440u0430u043du0441u043au0440u0438u043fu0442u0430u0437u0443 virus of avian u043cu0438u0435u043bu043eu0431u043bu0430u0441u0442u043eu0437u0430 (amv - rt).;10. way to 1, where the use of dna selected from the group, u0441u043eu0441u0442u043eu00a0u0449u0435u0439 of genome dna, the dna u043bu00a0u043cu0431u0434u0430 - u0444u0430u0433u0430, u043fu043bu0430u0437u043cu0438u0434u043du043eu0439 dna dna dna dna u043cu043bu0435u043au043eu043fu0438u0442u0430u044eu0449u0438 u0431u0430u043au0443u043bu043eu0432u0438u0440u0443u0441u043eu0432, plant and x.;11. method for 1, where u0434u043bu00a0 dual u0440u0430u0441u0449u0435u043fu043bu0435u043du0438u00a0 nucleic acid using more than one u0440u0435u0441u0442u0440u0438u043au0446u0438u043eu043du043du043eu0439 endonuclease.;12. method for 1, where u0434u043bu00a0 dual u0440u0430u0441u0449u0435u043fu043bu0435u043du0438u00a0 use u0440u0435u0441u0442u0440u0438u043au0446u0438u043eu043du043du044bu0435 endonuclease EcoRI and Hind iii.;13. way to 1, where the microwave radiation can be conducted in any device or in any cell in which microwave radiation can be generated.;14. way on the way to 1, which can be partially or fully automated using a specially designed device.