Balkan Journal of Medical Genetics

FREQUENCIES OF THE MEFV GENE MUTATIONS IN AZERBAIJAN
Huseynova LS1, Mammadova SN, Aliyeva KAA
*Corresponding Author: Lala S. Huseynova, Ph.D., Department of Medical Biology and Genetics, Azerbaijan Medical University, Anvar Gasimov Street 14, Baku City AZ1022, Azerbaijan. Tel.: +994- 506-630-623. E-mail: royahuseynova2006@gmail.com
page: 33

MATERIALS AND METHODS

All molecular genetic methods for detecting mutations are based on differences in the DNA sequence. Material used was venous blood with anticoagulant of 42 patients in 2016-2021. The age of the studied patients ranged from 2 to 29 years old. Genome DNA was obtained by automatic isolation from 200 μL of venous blood. The DNA concentration was measured by the Digital spectrometer. The integrity of the isolated genomic DNA was detected in a 2% agarose gel. The venous blood for research was drawn into a tube containing EDTA or heparin. Genomic DNA and RNA kits made by Qiagen GmbH (Hilden, Germany) were used for analysis. Integrity and quantity of genomic DNA and polymerase chain reaction (PCR) products were identified by electrophoresis on 2% agarose gel (PowerPacBasicGelDoc™ EZ; Bio-Rad Laboratories, Hercules, CA, USA). The genome DNA underwent the PCR procedure for every protein-encoding exon of the MEFV gene. Positive PCR samples that were checked by electrophoresis in agarose gel were purified by an enzymatic method. Purified product was dyed with fluorescent dye by BiqDye Terminator V.3.1. (Applied Biosystems, Foster City, CA, USA) and processed by Cycle Sequencing PCR. Positive Cycle Sequencing PCR samples, controlled by electrophoresis in agarose gel, were extracted from the BiqDye XT (Applied Biosystems with dye-purifying agent. (Figure 1 and Figure 2). Polymerase chain reaction was carried out in a following conditions: denaturation at 96 °C for 30 seconds; annealing at 55 °C for 30 seconds; extension at 75 °C for 1 min. This cycle was repeated 25 times, 72 °C for 10 min. and 4 °C pause. The PCR was carried out on a Professional Thermocycler Biometra system (Biometra Biomedizinische Analytik GmbH, Göttingen, Germany). A pair of for-ward and reverse primers was used for each genomic fragment. For the purification of DNA fragments after the first stage of PCR, a set of magnets was used: Agencourt AMPure XP PCR purification and SPRIPlate 96 Super Magnet Plate (Beckman Coulter Inc., Beverly, CA, USA). The second amplification of the purified DNA fragments was carried out in the following condition: denaturation at 95 °C for 30 seconds; annealing at 55 °C for 30 seconds; extension at 77 for 2 min. This cycle was repeated 25 times, and 72 °C for 10 min. and 4 °C pause. The nucleotide sequence of purified fragments was studied in GENOME Lab GeXP™ Sequencing (SCIEX, Brea, CA, USA). The obtained nucleotide chains were identified through SeqScape® version 2.7 software program (Applied Biosystems, Foster City, CA, USA; http:/tools.thermofisher. com/content/sfs/manuals/4401740.pdf), then compared by means of the National Center for Biotechnology Information (NCBI) Blast Ce, to normal MEFV nucleotide chains, and only then were the substitutions and mutations identified. Two DNA fragments were amplified: in exon 2, 360 nucleotide bases long and in exon 10, 400 nucleotide bases long. We used primers for exon 2 (forward): 5’-AAA ACG GCA CAG ATG ATT CCG-3’ and (reverse): 5’-AAG GGC CTG CAC TCC TTC-3’; and for exon 10 (forward): 5’-AGC AGG AAG AGA GAT GCA GTG-3’ and (reverse): 5’-TTG GAG ACA AGA CAG CAT GG-3’.

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