BssHII (BsePI) restriction enzyme

Protocol tips

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	The restriction enzymes used were AluI (TaKaRa) for Glu178Glu (reference SNP no. rs45483293) BssHII (TaKaRa) for Ala196Ala (rs5480) BsmAI (New England BioLabs, Beverly, MA, USA) for Ser180Phe (found in a Japanese patient with apparent mineralocorticoid excess) and HhaI (TaKaRa) for Arg208His (rs28934592) within exon 3 and HhaI for Arg279Cys (rs28934594) within exon 5. Among these four polymorphisms, the HapMap frequency for JPT is available only with Ala196Ala and is reported as 0.

Protocol tips

The restriction enzymes used were AluI (TaKaRa) for Glu178Glu (reference SNP no. rs45483293) BssHII (TaKaRa) for Ala196Ala (rs5480) BsmAI (New England BioLabs, Beverly, MA, USA) for Ser180Phe (found in a Japanese patient with apparent mineralocorticoid excess) and HhaI (TaKaRa) for Arg208His (rs28934592) within exon 3 and HhaI for Arg279Cys (rs28934594) within exon 5. Among these four polymorphisms, the HapMap frequency for JPT is available only with Ala196Ala and is reported as 0.

Publication protocol

"To search for frequent polymorphisms in HSD11B2, we employed the nonradioisotopic PCR followed by single-strand conformational polymorphism(SSCP) and the RNase cleavage assay. In PCR-SSCP for each exon 2–5, 50 ng of genomic DNA was subjected to PCR as described previously,2 and the reaction was electrophoresed in 0.5 MDE gels (FMC Bio Products, Rockland, ME, USA) and visualized with silver staining. To screen polymorphisms in the region from the proximal promoter (nt 679) to 50 -intron 1 (PV) and theregion from 30
-intron 1 to 30 -UTR (WZ), we employed the non-isotopic RNase cleavage assay. The 961-bp-long PV segments were amplified using a sense primer located in the promoter region (50 -TGT CCC AGG CAG GTT
TTG TGG-30; nucleotides (nt) 730 to 710) and an antisense primer in intron 1 (50-CCC TCG AGC CTG GAG TCC-30), followed by nestic PCR usinga sense T7-PV primer (50-TAA TAC GAC TCA CTA TAG GGG TGA GCG
CGC CTT-30; to nt 679) and an antisense SP6-PV primer (50-ATT TAG GTGACA CTA TAG GAG TCC CCG CGC TCC-30; from 50-intron 1 þ 18 nt). Thesupplied buffer was used at a concentration of 1.5 mM Mg2 þ with 2.5 Uml1
of PLATINUM pfx DNA polymerase (Gibco-BRL, Gaithersburg, MD, USA).After initial denaturation at 96 1C for 2 min, a manual hot-start at 80 1C was employed, followed by 24 cycles (1st PCR) and 26 cycles (nestic PCR) of 96 1C for 25 s and 68 1C annealing/extension for 90 s. The 1438-bp-long WZ segments were amplified using a sense primer in intron 1 (50-CTG CTGGTG GCT TGG TTT G-30) and an antisense primer in the 30-untranslated
region (50-ATC GTA ATG CTG GGG GTT TTC-30), followed by nestic PCRusing a sense T7-WZ primer (50-TAA TAC GAC TCA CTA TAG GGT GATTCT GGG GTT GTC-30; to 30-intron 1 54 nt) and an antisense SP6-WZ
primer (50-ATT TAG GTG ACA CTA TAG AAT GGC TGG GCC ATA GGTG-30; from þ 9 nt from stop codon). The supplied buffer was used at aconcentration of 1 mM Mg2 þ with 5 Uml1 of Pyrobest DNA polymeraseaKaRa, Osaka, Japan). After initial denaturation at 96 1C for 2 min, a manual hot-start at 80 1C was employed, followed by 24 cycles (1st PCR) and 26 cycles (nestic PCR) of 96 1C for 15 s, 65 1C annealing for 20 s, and 72 1C extension for 90 s. The in vitro transcription of the PCR products, the subsequent hybridization and the RNases digestion were completed following the manufacturer’s protocol for a MisMatch Detect II kit (Ambion, Austin, TX,
USA). The screenings were examined in 40 normal subjects, 10 patients with essential hypertension and 5 patients with pseudoaldosteronism. In addition, several reported polymorphisms or mutations were explored in an additional
80 normal subjects and 20 patients under hemodialysis resulting from various pathological origins by PCR-RFLP. The restriction enzymes used were AluI (TaKaRa) for Glu178Glu (reference SNP no. rs45483293),10,14 BssHII (TaKaRa) for Ala196Ala (rs5480),15 BsmAI (New England BioLabs, Beverly, MA, USA) for Ser180Phe (found in a Japanese patient with apparent mineralocorticoid excess)16 and HhaI (TaKaRa) for Arg208His (rs28934592) within exon 3 and HhaI for Arg279Cys (rs28934594)17 within exon 5. Among these four polymorphisms, the HapMap frequency for JPT is available only with Ala196Ala and is reported as 0"

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