Bigdye terminator v1.1 cycle sequencing kit

PCR products from DNA and cDNA, were purified using a Qiaquick PCR purification Kit (Qiagen TM, ME, DE) and bidirectionally sequenced using a BigDye_Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA) following manufacturer’s recommendations. Sequences were dye-terminator removed by DyeEx_ 2.0 spin kit (Qiagen TM, ME, DE) and run on a 3500 Genetic Analyzer (Applied Biosystems, CA, USA). Electropherograms were analyzed using Sequencing analysis v5.2 and sequence scanner v1.0 softwares (Applied Biosystems, CA, USA). The sequences obtained were compared to others in GenBank using the BLAST program.

PCR products from DNA and cDNA were purified using a Qiaquick PCR purification Kit (Qiagen TM, ME, DE), and bidirectionally sequenced using a BigDye_Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA) following manufacturer’s recommendations. Sequences were dye-terminator removed by DyeEx_2.0 spin kit (Qiagen TM, DE) and run on a 3500 Genetic Analyzer (Applied Biosystems, CA, USA). Electropherograms were analyzed using Sequencing Analysis v5.2 and Sequence Scanner v1.0 software (Applied Biosystems, CA, USA). The sequences obtained were compared to others in GenBank using the BLAST program.

Genomic sequences of KCNH1, STK36, and ZNF517 were obtained from UCSC Genome Browser (December 2013). A flanking region around each sequence variant site was amplified by PCR with the following primer pairs: forward primer (5′-TCAACGCTTTTGAGAACGTG-3′) and reverse primer (5′-TGTCTTGGTGTCCTCGTCAA-3′) for KCNH1 (NM_002238); forward primer (5′-CATCCCTCATCTCTGGCCTG-3′) and reverse primer (5′-ACTTTTACCTTGCCCTGAATCA-3′) for STK36 (NM_001243313); and forward primer (5′-TTCAAGCAAAGCTCCATCCT-3′) and reverse primer (5′-GGTGTGGAACTTCTGGTGCT-3′) for ZNF517 (NM_213605). Primers for the PCR amplifications were designed using Primer3 Software. PCR reactions were performed using Taq DNA polymerase (Invitrogen Life Technologies, Carlsbad, CA, USA). PCR fragments were run on 1 % agarose gel. The fragments were purified using the Illustra_ GFX_ PCR DNA and Gel Band Purification Kit (GE Healthcare) and then sequenced using the Big Dye_ Terminator v 1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Sequence reaction was purified on Sephadex G50 (Amersham Pharmacia Biotech, Foster City, CA), and then loaded into an ABI 3100 system after the addition of Hidi formamide. Electropherograms were analyzed using Sequence Analysis Software version 5.2 (Applied Biosystems) and then aligned with the reference sequences using ChromasPro version 1.22 (Technelysium, Queensland, Australia).

The genomic sequence of PCDH19 (NM_001184880.1) was obtained from UCSC Genomic Browser. Primers used for PCR amplification were designed using Primer3 software (http://frodo.wi.mit.edu accessed on 30 November 2022) to amplify exon 6 of the PCDH19 gene, including the p.Asp1024Asn detected by WES in the patient B. PCR reactions were performed using Taq DNA polymerase (Invitrogen Life Technologies, Carlsbad, CA, USA). PCR fragments were run on 1% agarose gel. The fragments were purified using a Sigma-Aldrich kit and then sequenced using the Big Dye_Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). The sequence reaction was purified on a Sephadex G50 (Amersham Pharmacia Biotech, Foster City, CA, USA) and then loaded into an ABI3500 system after the addition of Hidi formamide. Electropherograms were analyzed using Sequence Analysis Software version 5.2 (Applied Biosystems, Foster City, CA, USA) and then aligned with the reference sequences using ChromasPro v1.7.6.1 (Technelysium, Queensland, Australia).

Positive clones (defined as PCR fragments larger than the estimated length product of the wild‐type FLT3 fragment) were re‐amplified using a forward primer without the 6FAM label and further purified using ExoZap‐IT (Applied Biosystems), or illustra Exoprostar 1‐step (VWR, Radnor, PA, USA) and PCR‐amplified for sequencing. BigDye v1.1 Terminator cycle sequencing kit (Applied Biosystems) was used to perform direct sequencing and the products were analysed on an ABI 3730 Genetic Analyzer (POP7 polymer), (Applied Biosystems) according to the manual. The sequences were analysed using FinchTV (Geospiza Inc., Seattle, WA, USA).