Abi prism bigdye terminator sequencing kit v1

Sequencing analysis was performed on positive amplicons obtained via DHPLC. PCR reactions were performed using Taq Gold Polymerase^® (Applied Biosystems, Foster City, CA). The sizes of PCR products were verified by electrophoresis on a 2% agarose gel. PCR products were purified using ExoSAP‐IT^® (USB Corporation, Cleveland, OH) according to the manufacturer's protocol and were sequenced in both directions using an ABI PRISM BigDye terminator sequencing kit v1.1 (Life Technologies) on an ABI Prism 3130 Genetic Analyzer (Life Technologies). Primers used for amplification and sequencing were the same as those used for DHPLC analysis. Bidirectional DNA sequences were compared to normal DNA and to NM_000267.3 reference sequence (NC_000017.10). NF1 exon numbering followed that used in the NCBI Reference Sequence (NG_009018.1) and included exons 1 to 58 (skipping exon 31, formerly referred to as exon 23a). Family members were usually studied by direct sequencing for the known mutation only.

The MethPrimer tool (http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi) was used to predict the CpG Island based on the promoter sequence provided from UCSC database, and BSP primers were designed according to the standard parameters and synthesized by GenFanAvaran Co. (Tehran, Iran). The PCR reactions were performed using 12 μl of Taq DNA Polymerase 2x Master Mix RED with 2 mM MgCl2 final concentration (Ampliqon, Odense M, Denmark); 2 μl bisulfite treated DNA (approximately 50 ng), 0.5 μl of each forward and reverse primers (10pM), and ddH2O to the final volume of 25 μl. The reactions were conducted in a SimpliAmp thermal cycler device (Applied Biosystems) with a primary denaturation for 5 min at 95 °C followed by 40 cycles of denaturation at 95 °C for 30 Sec, annealing temperature with descending touchdown system (− 0.2 °C) starting at 59 °C for 45 Sec, an extension at 72 °C for 45 Sec; and a final extension at 72 °C for 10 min. Following DNA cleanup procedure using the shrimp alkaline phosphatase enzyme for PCR product purification (Thermofisher Scientific). The products were then sequenced with an ABI PRISM BigDye terminator sequencing kit v1.1 (Life Technologies), and directly analyzed by an automated ABI 3130 Genetic Analyzer (Life Technologies).

The PCR products were sequenced with an ABI PRISM BigDye terminator sequencing kit v1.1 (Life Technologies) and directly analyzed by an automated ABI 3130 Genetic Analyzer (Life Technologies).