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METHOD FOR PRODUCING THE UNITED DNA fragments from complex mixtures of high molecular DNA ( 'SPLICE' and translocation of DNA IN VITRO)

机译:从高分子DNA的复杂混合物(“ SPLICE”和体外DNA易位)生产UNITED DNA片段的方法

摘要

FIELD: biotechnologies.; SUBSTANCE: the invention is referred to the field of biotechnology. Performance of two PCR rounds is enough for obtaining of the end product. In the first round primary DNA fragments are obtained using multiplexed PCR, in the second round cohesive ends appear in the obtained DNA fragments, fragments fuse and the resulting product is amplified. Aggregate of high-molecular weight DNA or human genomic DNA can be used as primary material in the first round, PCR products from the first round are used in the second round. The technique allows for obtaining of rather precise fragments consisting of exons alone. If there is no necessity for obtaining of compound fragments without inserts, it is proposed to get compound fragments from the aggregate of high-molecular weight DNA in one PCR reaction. Multiplexed DNA using special primers that contain ends that are unique for genomic DNA 5' and that are mutually complementary, by which formation of cohesive ends is achieved in synthesized primary fragments. In this process a pair of primers flanking the common compound fragment, which are fully complementary with external edges of desired compound DNA fragment and intended for its amplification, is used, as well.; EFFECT: fusion of PCR fragments that do not require intermediate treatment, preliminary cloning of primary DNA fragments, and also separate amplification of primary fragments. ;2 cl, 7 dwg, 3 ex
机译: FIELD:生物技术。 物质:本发明涉及生物技术领域。进行两次PCR反应足以获得最终产物。在第一轮中,使用多重PCR获得初级DNA片段,在第二轮中,获得的DNA片段中出现内聚末端,片段融合并扩增所得产物。高分子量DNA或人类基因组DNA的聚集体可以在第一轮中用作主要材料,在第一轮中使用来自第一轮的PCR产物。该技术允许获得仅由外显子组成的相当精确的片段。如果不需要获得没有插入片段的化合物片段,建议在一次PCR反应中从高分子量DNA的聚集物中获得化合物片段。使用特殊引物的多重DNA,该引物的末端对于基因组DNA 5'而言是唯一的,并且相互互补,由此可以在合成的一级片段中形成内聚末端。在该方法中,还使用了在共同化合物片段侧翼的一对引物,它们与所需化合物DNA片段的外边缘完全互补,并打算用于其扩增。 效果:不需要中间处理的PCR片段的融合,初级DNA片段的初步克隆以及初级片段的单独扩增。 ; 2 cl,7 dwg,3 ex

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