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URL: https://dx.doi.org/DOI:10.17504/protocols.io.2v9ge96

Authors: Predrag Noveski, Marija Terzic, Marija Vujovic, Maja Kuzmanovska, Emilija Sukarova Stefanovska, Dijana Plaseska-Karanfilska

Summary: Quantitative fluorescent polymerase chain reaction (QF-PCR) is reliable and efficient method for the rapid prenatal diagnosis of common fetal aneuploidies. In this protocol we first describe a procedure for isolation of DNA from amniocytes and chorionic villi with the use of commercial kit intended for the isolation of nucleic acids from viruses, which, taking into account the phenomenon of increased fragmentation of the DNA material of fetal origin, proved to be appropriate for prenatal diagnosis. (Section 1). As part of the isolation procedure, we also include a procedure for treating amniotic fluid with visible contamination (blood with possible origin from the mother).Тhe presented procedure consists of simple physical separation of the maternal blood cells from the fetal material in order to reduce the failure rate due to maternal cell contamination. .justify:after { content: ""; display:inline-block; width: 100%; } In the second part of the protocol (Section 2) we are presenting procedure for performing the QF-PCR analysis. Here, we describe an in-house one-tube multiplex QF-PCR method including 20 PCR markers (15 STR markers and 5 fixed size) for rapid prenatal diagnosis of chromosome 13, 18, 21, X and Y aneuploidies, which targeted in total 26 genomic positions. Of them, three markers (D13S258, D13S305 and D13S1817) were for analysis of the aneuploidies for the chromosome 13, four (D18S386, D18S390, D18S391 and D18S535) for the chromosome 18, four (D21S1411, D21S1414, D21S1435 and D21S1446) for the chromosome 21 and six for the analysis of the sex chromosome aneuploidies. Two of the sex chromosome markers (DXS6803 and XHPRT) amplified sequences only on chromosome X (for counting chromosome X), two (AMELX/Y and DXYS218) co-amplified sequences of both X and Y chromosomes (for counting chromosomes X and Y in male samples and chromosome X in female samples), one (TAF9B) was for co-amplification of sequences on chromosomes X and 3 (for counting chromosome X) and one (SRY) located on chromosome Y was for male sex determination. Three remaining markers (MYPT2/Y, DYS448 and CDY1/2) were used simultaneously for detection of the Y chromosome aneuploidies and microaberrations in the azoospermia factor region “c” (AZFc), located on the Y chromosome. .justify:after { content: ""; display:inline-block; width: 100%; } For the cases with inconclusive results, we used additional QF-PCR reactions specific for given chromosome (Section 3). For chromosomes 13, 18 and 21 the primers were combined in one multiplex reaction each: four STR markers for chromosome 13 (D13S742, D13S628, D13S634 and D13S631), three STR markers for chromosome 18 (D18S51, D18S1367 and D18S978) and four STR markers for chromosome 21 (D21S1437, D21S11, D21S1412 and D21S1441). For the sex chromosomes we have used three markers as separate PCR reactions (for DXS6809, DXS996 and X22) and additionaly if needed, previously published multiplex reaction [File 1] for detection of sex chromosome aneuploidies in infertile patients. .justify:after { content: ""; display:inline-block; width: 100%; }

Associated Publications: Noveski P, Terzic M, Vujovic M, Kuzmanovska M, Stefanovska ES, Plaseska-Karanfilska D (2019) Multilevel regression modeling for aneuploidy classification and physical separation of maternal cell contamination facilitates the QF-PCR based analysis of common fetal aneuploidies. PLoS ONE 14(8): e0221227. doi: 10.1371/journal.pone.0221227

Affiliations: Research Center for Genetic Engineering and Biotechnology “Georgi D. Efremov”, Macedonian Academy of Science and Arts, Skopje, Republic of Macedonia, Research Center for Genetic Engineering and Biotechnology “Georgi D. Efremov”, Macedonian Academy of Science and Arts, Skopje, Republic of Macedonia, Research Center for Genetic Engineering and Biotechnology “Georgi D. Efremov”, Macedonian Academy of Science and Arts, Skopje, Republic of Macedonia, Research Center for Genetic Engineering and Biotechnology “Georgi D. Efremov”, Macedonian Academy of Science and Arts, Skopje, Republic of Macedonia, Research Center for Genetic Engineering and Biotechnology “Georgi D. Efremov”, Macedonian Academy of Science and Arts, Skopje, Republic of Macedonia, Research Center for Genetic Engineering and Biotechnology “Georgi D. Efremov”, Macedonian Academy of Science and Arts, Skopje, Republic of Macedonia

External URL: https://doi.org/10.1371/journal.pone.0221227

Version: 1

Publication Date: 2019

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