Balkan Journal of Medical Genetics

CLINICAL AND MOLECULAR DATA ON MENTAL RETARDATION IN BULGARIA
Todorov T1#, Todorova A1#, Avdjieva D2, Dimova P3, Angelova L4, Tincheva R2 and Mitev V1
*Corresponding Author: Tihomir Todorov, Department of Medical Chemistry and Biochemistry, Sofia Medical University, 2 “Zdrave” str., Sofia 1431, Bulgaria; Tel./Fax: +359 29530715; tisho.todorov@abv.bg
page: 11

PROTOCOLS FOR MECP2, CDKL5 and ARX GENE ANALYSIS

Sequencing. These X-chromosome genes were directly sequenced, as all the exons (including the first non coding one for the MECP2 gene) and intron/exon splice site borders were included/ covered. The sequence of primers used for PCR amplification was taken from the literature [6,12,13] or designed in our laboratory (the primer sequence is available upon request). Standard PCR conditions were applied: primer concentration 0.4 μM, 0.2 mM dNTPs, 0.4 M Betaine (Sigma, St. Louis, MO, USA), 4% DMSO; 1× supplied PCR reaction buffer (Genet Bio, Chungnam, Korea) and 0.5U Prime Taq (Genet Bio). The annealing temperature varied between 55°C and 68°C. The PCR products were purified by PCR Product Pre-Sequencing Kit (USB, Affymetrix, Inc., Santa Clara, CA, USA) containing 4 U exonuclease I (10 U/μL) and 0.8 U shrimp alkaline phosphatase (2 U/μL). The sequencing reaction was performed by ABI PRISM™ BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems) and analyzed on an ABI PRISM™ 310 Genetic Analyzer (Applied Biosystems). MLPA analysis. The SALSA MLPA P015-D1 MECP2 probemix was used to screen for large deletions/ duplications along the genes MECP2, CDKL5 and ARX. The analysis was performed following the manufacturer’s instructions [14]. Fifty to 200 ng of DNA was diluted in TE buffer to a total volume of 5 μL. The diluted samples were subjected to hybridization with the probes specific for the MECP2, CDKL5 and ARX genes, and 17 control probes. Hybridization was performed overnight at 60°C. Hybridized probes were ligated by a specific ligase mix, provided by the manufacturer. The final step represents PCR amplification of the obtained ligated fragments. The PCR buffer, PCR primers (6- FAM labeled), the enzyme dilution buffer and the polymerase were provided in the kit. The obtained PCR products were analyzed on an ABI PRISM™ 310 Genetic Analyzer (Applied Biosystems) in the presence of ROX500 size standard (Applied Biosystems). Each patient sample was analyzed simultaneously with at least two normal controls. The relative peak ratios in order to assess deletions were calculated using the formula: r = (peak areapatient/mean peak area both neighbouring control peakspatient)/(peak areacontrol/mean peak area both neighbouring control peakscontrol). Relative ratio of <0.6 corresponded to a deletion.

Number 27 VOL. 27 (2), 2024	Number 27 VOL. 27 (1), 2024
Number 26 Number 26 VOL. 26(2), 2023 All in one	Number 26 VOL. 26(2), 2023
Number 26 VOL. 26, 2023 Supplement	Number 26 VOL. 26(1), 2023
Number 25 VOL. 25(2), 2022	Number 25 VOL. 25 (1), 2022
Number 24 VOL. 24(2), 2021	Number 24 VOL. 24(1), 2021
Number 23 VOL. 23(2), 2020	Number 22 VOL. 22(2), 2019
Number 22 VOL. 22(1), 2019	Number 22 VOL. 22, 2019 Supplement
Number 21 VOL. 21(2), 2018	Number 21 VOL. 21 (1), 2018
Number 21 VOL. 21, 2018 Supplement	Number 20 VOL. 20 (2), 2017
Number 20 VOL. 20 (1), 2017	Number 19 VOL. 19 (2), 2016
Number 19 VOL. 19 (1), 2016	Number 18 VOL. 18 (2), 2015
Number 18 VOL. 18 (1), 2015	Number 17 VOL. 17 (2), 2014
Number 17 VOL. 17 (1), 2014	Number 16 VOL. 16 (2), 2013
Number 16 VOL. 16 (1), 2013	Number 15 VOL. 15 (2), 2012
Number 15 VOL. 15, 2012 Supplement	Number 15 Vol. 15 (1), 2012
Number 14 14 - Vol. 14 (2), 2011	Number 14 The 9th Balkan Congress of Medical Genetics
Number 14 14 - Vol. 14 (1), 2011	Number 13 Vol. 13 (2), 2010
Number 13 Vol.13 (1), 2010	Number 12 Vol.12 (2), 2009
Number 12 Vol.12 (1), 2009	Number 11 Vol.11 (2),2008
Number 11 Vol.11 (1),2008	Number 10 Vol.10 (2), 2007
Number 10 10 (1),2007	Number 9 1&2, 2006
Number 9 3&4, 2006	Number 8 1&2, 2005
Number 8 3&4, 2004	Number 7 1&2, 2004
Number 6 3&4, 2003	Number 6 1&2, 2003
Number 5 3&4, 2002	Number 5 1&2, 2002
Number 4 Vol.3 (4), 2000	Number 4 Vol.2 (4), 1999
Number 4 Vol.1 (4), 1998	Number 4 3&4, 2001
Number 4 1&2, 2001	Number 3 Vol.3 (3), 2000
Number 3 Vol.2 (3), 1999	Number 3 Vol.1 (3), 1998
Number 2 Vol.3(2), 2000	Number 2 Vol.1 (2), 1998
Number 2 Vol.2 (2), 1999	Number 1 Vol.3 (1), 2000
Number 1 Vol.2 (1), 1999	Number 1 Vol.1 (1), 1998

About the journal ::: Editorial ::: Subscription ::: Information for authors ::: Contact