Treatment and Hemodynamic Characteristics
of the Patients.*
Supplementary Appendix 2
Tests for Mutations in BMPR2
The various exons of the gene for bone morphogenetic protein receptor 2 (BMPR2) were amplified with use of the polymerase chain reaction (PCR) from 100 ng of genomic DNA purified from paraffin-embedded tissue (Epicentre Technologies) with use of the proofreading Herculase polymerase (Stratagene) and 100 ng of each oligonucleotide primer (Deng). The PCR programs were optimized for each amplicon. Restriction-fragmentlength polymorphism (RFLP) analysis was performed by digesting the various amplicons with their specific restriction enzyme (New England Biolabs) and subjected to electrophoresis in NuSieve agarose at different concentrations in TBE buffer (TRIS base, boric acid, and EDTA) at 70 V. The gels were stained with ethidium bromide, and the images were analyzed with Kodak version 3.6 software to determine the molecular weights of the products of digestion. The editing of sequences and the construction of restriction maps were done with use of DNASTAR version 4.0 software.
Exons 3, 4, 8, 12b, and 12d were amplified by two rounds of touchdown PCR programs (programs in which the annealing temperature is gradually reduced in order to ensure amplification) as follows: a first round of 20 cycles at 94°C for 15 seconds and then 65°C for 30 seconds, with the temperature decreased by 2°C for each cycle, followed by 10 cycles at 94°C for 15 seconds and then 55°C for 30 seconds, and a second round of 20 cycles at 94°C for 15 seconds and then 60°C for 30 seconds, with the temperature decreased by 2°C for each cycle, followed by 25 cycles at 94°C for 15 seconds and then 60°C for 30 seconds. Exons 9, 6, and 12 were amplified with PCR consisting of denaturing at 92°C for 2 minutes and 35 cycles at 92°C for 30 seconds; amplification at 60.2°C, 61.9°C, and 59°C, respectively, for 30 seconds and at 72°C for 30 seconds; and finally 10 minutes of extension at 72°C.
