Combined Isothermal DNA Amplification Method (HDA–RCA) to Increase Analytical Sensitivity

Objective: Rolling circle amplification (RCA) is a sensitive and specific method of isothermal amplification of nucleic acids. RCA from the genomic DNA is complicated by the fact that the template target is present in the form of a long double-stranded DNA molecule that is difficult to access for ligase. One of the ways to prepare DNA for RCA analysis is the pre-amplification of genomic DNA by PCR, which leads to the formation of shorter products (amplicons) from which the ligation of the detecting oligonucleotide “padlock probe” is performed. The disadvantage of this method is the pre-amplification with thermal cycling (multiple melting of duplexes), which requires specialized equipment. Methods: A combined HDA-RCA approach is proposed to increase the sensitivity of RCA analysis in the study of full-genomic double-stranded DNA samples. Results and Discussion: It has been demonstrated that the use of pre-amplification increases the sensitivity of RCA in genomic DNA analysis. The PCR protocols are well reproducible, however, pre-amplification by PCR requires thermal cycling. With the HDA, it is possible to carry out pre-amplification in an isothermal mode, facilitating the unification of all stages into one, that is, the creation of a “one-pot” system for genomic DNA analysis using the RCA method. Conclusions: The possibility of using the proposed combined HDA-RCA method for detecting genome DNA in a sample is demonstrated. It has been demonstrated that pre-amplification using HDA increases the sensitivity of the RCA analysis, opening up the possibility of developing test systems for point-of-care diagnostics and in a single test tube without transferring material (“one-pot”).