Objective: For many years it has been known that free DNA of various origin is released into peripheral blood. An extremely short halftime of plasma DNA and many pre-analytical variables may affect the number of free DNA molecules in plasma specimens. The goal of our study was to compare six nucleic acids extraction protocols to define the optimal pre-analytical approach for further molecular analyses based on plasma DNA. Material and Methods: Extraction of nucleic acids (NA) from 900 μl of the aliquoted pool were performed by phenol/ chloroform DNA extraction method after SDS and proteinase K lysis without or with addition of a polyacryl DNA carrier before lysis, salting-out protein precipitation, DNA extraction with Qiagen spin microcolumns without or with the carrier addition, and a guanidinium isothiocyanate-based RNA extraction method. Extracts were characterized spectrophotometrically, electrophoretically, and according to the PCR amplificability. Results: Spectrophotometric analysis shows that the guanidinium and phenol/chloroform extraction methods provide the highest yields of nucleic acids. The mean yield values from 900 μl of plasma were 2.4 μg and 1.4 μg, respectively. The lowest yield was reached using commercial microcolumns without (0.4 ± 0.1 μg) or with the DNA carrier (0.2 ± 0.1 μg). However, fluorescence intensities of the extracts obtained by the phenol/chloroform procedure (with addition of the carrier) and both the microcolumn-based methods manifested very similar values. Examining by crossing-point analysis, microcolumn methods revealed the lowest number of cycles necessary to reach the exponential part of amplification. No fluorescence was detected in the guanidinium RNA extracts. Conclusion: Our data report that microcolumn extraction techniques are very suitable for preparation of plasma DNA specimens. Addition of the polyacryl DNA carrier does not seem to significantly influence the extraction yield.

        Key words: DNA; genomic DNA, free-cell DNA, extraction, nucleic acids, plasma, PCR, pre-analytical phase, spectrophotometry, electrophoresis.