Pop7 polymer

The presence of the VNTR-MNS16A TERT gene polymorphism was assessed in BC patients and in healthy women by PCR amplification followed by electrophoresis in sequencing gel, as described by Wysoczanska et al. [41 (link)]. PCR was performed in a 2720 Thermal Cycler instrument (Applied Biosystems, Foster City, CA, USA) using the forward and reverse primer sequences (5′-AGGATTCTGATCTCTGAAGGGTG-3′ and 5′-TAMRA-TCTGCCTGAGGAAGGACGTATG-3′) prepared by Genomed (Warsaw, Poland). The amplification procedure included an initial denaturation step for 5 min at 95 °C, followed by 35 cycles: 30 s at 95 °C, 30 s at 65 °C, 30 s at 72 °C and a final extension step for 10 min at 72 °C. The PCR products were diluted with formamide and a GeneScan™500 ROX™ dye Size Standard (Applied Biosystems, Foster City, CA, USA). The samples were denatured at 95 °C for 5 min and analyzed on the 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) with an eight-capillary system filled with POP7 polymer (Applied Biosystems, Foster City, CA, USA). The alleles were identified using the GeneMapper software version 4.2 (Applied Biosystems, Foster City, CA, USA).

We analyzed all DMD cases for large deletions and large duplications using MLPA SALSA P034/P035 DMD kits (http://www.mrc-holland.com) following the manufacturer’s instructions. In brief, denaturation, hybridization, ligation, and amplification steps were performed on a DNA Engine Dyad thermal cycler (Bio-Rad Laboratories Inc., Hercules, CA). Finally, PCR amplification was performed using SALSA MLPA PCR primers labeled with the FAM dye. A mixture of 0.7 μl of PCR product, 0.2 μl of 600 LIZ GS size-standard, and 9.0 μl of Hi-Di formamide was incubated for 3 min at 86 °C and cooled at 4 °C for 2 min. The MLPA product mix was separated on a POP7 polymer (Applied Biosystems Inc., Life Technologies, Foster City, CA) at 60 °C with the setting of 1.6 kV for injection voltage, 18 s for injection time, 15 kV for run voltage, and 1800 s for run time.

Capillary sequencing was performed to confirm the obtained RNA-Seq coding sequence of the beaver placental AP. Briefly, an Enhanced Avian HS RT-PCR Kit (Sigma-Aldrich, St. Louis, MO, USA) was used to transcribe RNA (from the same samples that were used for RNA-Seq) to cDNA in two-step RT-PCR. To synthesize the first strand of cDNA, a mix of dNTPs and random hexamers were used as primers. Furthermore, target cDNA was amplified with specific primers (Table 3) designed by used GENEIOUS R7 and Oligo Calc [50 (link)] on AP sequence identified in RNA-Seq.
The obtained AP amplicons were electrophoresed, gel-out purified and used as cDNA templates for the capillary sequencing (3130 Genetic Analyzer, Applied Biosystems, Foster City, CA, USA) of both strands in sense and anti-sense directions. Labeling was performed with a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol. Labeled amplicons were purified with a BigDye X Terminator Purification Kit (Applied Biosystems, Foster City, CA, USA) and separated in capillaries filled with POP-7™ polymer (Applied Biosystems, Foster City, CA, USA). The identified beaver AP cDNA sequence was analyzed using GENEIOUS R7 software. Additionally, in silico analyses of the cDNA were performed with the following online tools [51 (link),52 (link),53 ].

All colonies were selected from the highest dilution PCA spread plates, which usually contained 30–100 isolates using an inoculation loop and sub‐culture at 30°C for 24–48 h. All colonies were isolated and purified using PrepMan ultra sample preparation reagents. Genomic DNA was diluted 1:500 in nuclease‐free water. Polymerase chain reaction (PCR) and cycle sequencing were performed according to the manufacturer's manual using the Fast MicroSeq 500 Bacterial Identification kit (Applied Biosystems) with a PCR machine (Biometra). Extension and sequencing clean‐up were performed with the ExoSAP‐IT (USB Products, Affymetrix, Inc.) and Performance DTR cartridges (Edge BioSystems), respectively, according to the manufacturer's instructions. Sequences were detected on a 3500xL Genetic Analyzer (Applied Biosystems) with POP‐7 polymer (Applied Biosystems). Analysis of sequencing data was performed using the MicroSEQ ID Software (Ver. 2.2).

For confirmation of plasmid constructs, DNA sequencing was done using the BigDye^® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Inc., United States) essentially as instructed by the manufacturer. The samples were run on an ABI3130 Genetic Analyzer using the pop-7 polymer (Applied Biosystems Inc.). DNA sequence data were compiled and analyzed by using the Chromas 1.45². DNA sequences were obtained from J. Craig Venter Institute (JCVI)³, and protein domain information was obtained from Kyoto Encyclopedia of Genes and Genomes (KEGG)⁴. BLASTN and BLASTP programs were used to search for homologous nucleotide or protein sequences, respectively, in the database⁵. Clustal Omega software⁶ was used for the alignment of protein sequences and further modified through GeneDoc software version 2.7.000⁷. For designing primers, Primer3 software⁸ was used. For designing qRT-PCR primers, Primer Express 3.0 software (Applied Biosystems, United States) was used.

The open reading frame of coding exon 2 of the GJB2 gene was amplified using primers and PCR conditions previously described [37 (link)]. Applied Biosystems™ BigDye™ Terminator v1.1 Cycle Sequencing Kit and clean-up (Thermo Fisher, Waltham, MA, USA) was used. Sequencing was performed on an ABI 3730 Genetic Analyzer—36 cm array and POP7 polymer (Applied Biosystems). Data analysis was performed using Mutation Surveyor^® DNA Variant Analysis Software v.5 (Softgenetics, State College, PA, USA). Variant classification followed American College of Medical Genetics and Genomics (ACMG) recommendations and consulted online databases ClinVar, Varsome [38 (link)]. The guidance ACMG developed on the interpretation of variants identified in Mendelian disorders recommends evidence-based classification of variants into five categories: ‘pathogenic’, ‘likely pathogenic’, ‘uncertain significance’, ‘likely benign’, and ‘benign’ [39 (link)]. Deafness Variation Database was consulted for pathogenicity calls at https://deafnessvariationdatabase.org (accessed on 16 October 2022) [40 (link)].