DOI: 10.24075/brsmu.2017-04-03

METHOD

Modified multiplex real-time PCR for quantification of differently sized cell-free DNA fragments

Sokolova EA1,2, Khlistun IV1, Kushlinsky DN3
About authors

1 Biocode Ltd., Moscow, Russia

2 Laboratory of Paleogenomics, Faculty of Natural Sciences,
Novosibirsk State University, Novosibirsk, Russia

3 N. N. Blokhin National Medical Research Center of Oncology, Moscow, Russia

Correspondence should be addressed: Ekaterina Sokolova
Novomereschensky proezd, d. 9, str. 1, Moscow, Russia, 119619; ur.xednay@8062aeavolokos

About paper

Contribution of the authors to this work: Sokolova EA — research planning, data analysis, drafting of a manuscript; Khlistun IV — experimental work, drafting of a manuscript; Kushlinsky DN — biomaterial sampling, clinical characterization of patients, drafting of a manuscript.

Received: 2017-08-25 Accepted: 2017-08-30
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There is evidence that size distribution of cell-free DNA (cfDNA) fragments can be diagnostically relevant. The present work describes a multiplex quantitative real-time polymerase chain reaction technique modified and validated by the authors to study the degree of cfDNA fragmentation in blood plasma. Based on the detection of Alu and hLINE-1 repeats, this technique employs fluorescent probes. We selected suitable primers and probes, optimized PCR conditions and estimated the dynamic range and sensitivity threshold of the assay. The modified PCR had a dynamic range of 6 logs, its efficiency being over 90 %. We demonstrated that cfDNA fragmentation index did not differ significantly between healthy women (n = 16) and women with stage III–IV ovarian cancer (n = 14). Therefore, further research on a larger sample is needed using electrophoretic cfDNA fractionation.

Keywords: cell-free DNA, cfDNA, multiplex quantitative PCR, Alu repeats, hLINE-1 repeats, primers, fluorescent probe

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