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Cutting out the amplicon with restriction endonuclease – a way to exclude the false results of RT PCR with TaqMan probe

Akishev A.G., Netesova N.A., Abdurashitov M.A., Degtyarev S.Kh.

Real-time PCR with TaqMan probe (RT PCR) is one of the main methods for analyzing the structure of human DNA and is widely used in medicine. In this work, PCR RT of 4 fragments of human genomic DNA (chr1: 90717178-90717300, chr9: 97464604-97464756, chr11: 65647215-65647307, chr17: 4560027-4560175) was performed with DNA preparations from human blood obtained both by the standard method of phenol deproteinization and isolated on columns. Preparations of the original DNA and DNA after a treatment with a restriction endonuclease that cuts out the amplified fragment were used in PCR. In the case of native DNA preparations purified by column, PCR of 3 from 4 DNA fragments provided an overestimated Cq value. In the case of the phenol purification, an overestimated Cq value for native DNA preparations was obtained for 2 from 4 DNA fragments. For DNA preparations obtained by the column purification, treatment of DNA with a restriction endonuclease that cuts out the amplified fragment leads to normal Cq values for all four analyzed DNA fragments. Thus, RT PCR of native DNA obtained by the column purification may result in an overestimation of Cq, which is avoided if DNA previously has been hydrolyzed by a restriction endonuclease that cuts out the amplicon.

Comparison of the substrate specificity of GlaI recombinant site-specific methyl-directed DNA endonuclease and the native enzyme isolated from Glacial ice bacterium strain

Valery A. Chernukhin, Vladimir S. Dedkov, Danila A. Gonchar, Murat A. Abdurashitov, Alexander G. Akishev, Tatyana N. Nayakshina, Elena N. Lomakovskaya and Sergey Kh. Degtyarev

cloning of GlaI site-specific methyl-directed DNA endonuclease gene and the comparison of the substrate specificity of recombinant and native enzymes is described. The analysis of recombinant GlaI endonuclease properties showed that the substrate specificity of native and recombinant enzymes didn't differ, but the concentration of recombinant enzyme exceeded 10 times of native enzyme

A new restriction endonuclease LmnI recognizes the nonpalindromic DNA sequence 5’-GCTCC(1/-1)-3’

Valery A. Chernukhin, Danila A. Gonchar, Murat A. Abdurashitov, Tatyana N. Nayakshina, Vladimir S. Dedkov, Natalya A. Mikhnenkova, Elena N. Lomakovskaya and Sergey Kh. Degtyarev

We have discovered a bacterial strain Lysinibacillus manganicus An22 that produces the new prototype of restriction endonuclease named LmnI. This enzyme recognizes a nonpalindromic DNA sequence 5’-GCTCC-3’/3’-CGAGG-5’. The LmnI restriction endonuclease preparation with the concentration of 1000 units/ml was isolated using four chromatographic steps. It was shown that new enzyme cuts its recognition sequence forming 3’-protruding ends as indicated by the arrows: 5’-GCTCCN↓-3’/3’-CGAG↑GN-5’, and can be applied to a IIS type of restriction-modification systems.