Abi prism 377 dna

The PCR products of the eight overlapping fragments for each Fennoscandian CPXVs were purified using GFX PCR DNA and Gel Band Purification Kit (GE Health, Uppsala, Sweden), following the manufacturers instruction. Cycle sequencing reactions were performed using Big Dye 3.1 Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Each purified DNA fragment was sequenced in both orientations and at least two independent DNA cycling reactions were performed for each fragment. The cycle sequencing extension products were electrophoresed using ABI Prism™ 377 DNA automatic sequencer (Applied Biosystems).

Genomic DNA of each participant was extracted from peripheral blood lymphocytes using the QIA amp DNA Blood Mini Kit (QIAGEN, Hilden, Germany). The proband underwent next-generation sequencing using a panel containing 101 monogenic hypertension-related genes (Supplementary Table 1). Candidate variants were predicted by in silico analysis using MutationTaster2 (http://www.mutationtaster.org) and were verified in the proband and his family members by PCR. Then these candidate variants were sought in 100 hypertensives and 100 healthy controls to evaluate whether the variations were common genetic polymorphisms. All PCR products were sequenced using an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA, USA).