The reason of non-specific background of methodologies for random mutation identification: X-structures formation in purified PCR products solutions

Abstract

Chemical or Enzyme Mismatch Cleavage represent the methodologies directed to point random mutation detection. They are based on the formation of heteroduplexes consisting of one wild-type and one mutant strand from PCR-products. During mutation detection analysis, high nonspecific background prompted a detailed investigation of the DNA used in the study. The phenomenon of diverse DNA structures of double size of PCR product approximately and of higher molecular weight appearing in concentrated solutions of purified DNA fragments was demonstrated for 5 PCR products differing both by size and sequence (various genes PCR products from 118 bp to 1249 bp). The present investigation on the model of p53 cDNA 1249 bp fragment confirm that the diverse DNA structures are formed by double stranded DNA fragments via Sl-nuclease analysis: treated by the enzyme DNA fragments retain the ability to form higher molecular weight structures. Using the number of following purification procedures it was demonstrated, that the phenomenon is due to interaction of purified ds DNA fragments and is not connected with the any contamination of standard PCR products. The electroellution of twice the size DNA structures from agarose gel followed by electron microscopy reveals the presence of X-structure in this fraction of DNA. In our investigation the ability of DNA to interact with formation of Holliday junction, the intermediate structures of the recombination process, without participation of cell proteins was demonstrated as it own phisico-chemical characteristic. Our data are significant for optimization of methodologies of CMC or EMC or other analysis using concentrated PCR product solutions via preventing of formation of complex DNA structures.