TABLE 1.
Oligonucleotide Primers, Polymerase Chain Reaction, and Denaturing High-Performance Liquid Chromatography Conditions for Mutational Analysis of Navβ Subunits
| Gene-Exon | Forward Primer (5′ to 3′) | Reverse Primer (5′ to 3′) | Size, base pairs | MgCl, mmol/L | Thermal Cycling Method† | Gradient 1, %B; Temp °C‡ | Gradient 2, %B; Temp °C |
|---|---|---|---|---|---|---|---|
| SCN1B-1 | CGC CTC TCG CCC CGC TAT TA | CTC CCG CCG CCC CGC CAG TG | 164 | 1* | 2 | 50.5 to 51.5; 67 | 48 to 58; 72 |
| SCN1B-2 | CCT GAC CTG AGC CTG CTG TC | TGC CCT CCC ATG CCG TTC C | 227 | 1* | 2 | 56.6 to 66.6; 65 | |
| SCN1B-3 | CCT TCC CCT CCC TGG CTA C | GGC AGG CAG CAC CCG ACT CA | 287 | 1* | 2 | 56.3 to 66.3; 63 | 52.6 to 62.6; 65 |
| SCN1B-4 | CAG CCT GGG CTA CCC CCT TA | CCC TGG GTG CCC TCC CAC CT | 220 | 1* | 2 | 53.7 to 63.7; 62.5 | |
| SCN1B-5 | CGG TCT GAT GAT GGG GTC AC | TTA CGG CTG GCT CTT CCT TG | 243 | 1* | 2 | 54.8 to 64.8; 63 | |
| SCN2B-1 | CCA TTC CTC CCT TGT AGT TCT | CCC CAT CCT CTT CAC ATT GC | 216 | 2 | 1 | 53.6 to 63.6; 61.5 | |
| SCN2B-2 | CCA ACA CTC CCA GGC ACA G | GAC CAG GGG CTT CAT GCC A | 316 | 2 | 1 | 57.2 to 67.2; 62 | 57.2 to 67.2; 63.5 |
| SCN2B-3 | GGC ATC CTC ACT GTC CTT G | AGG TGG GTG GGA AAG GTC A | 329 | 2 | 1 | 57.5 to 67.5; 63 | 57.5 to 67.5; 64 |
| SCN2B-4 | CAC GGG TAG TGG GGT GAT G | CGA GCA GGC AGG GTC ACT G | 346 | 2 | 1 | 57.9 to 67.9; 63.5 | |
| SCN3B-2 | GCA GTC CTT GAC CGA GGG A | AGA GGC AAG CCA GCC AGA G | 223 | 2 | 1 | 53.9 to 63.9; 59.5 | 53.9 to 63.9; 61.5 |
| SCN3B-3 | CCT CCC TCC TTC TTC TCC AA | CAG GAG CCA GGC TGG GAA C | 316 | 2 | 1 | 57.2 to 67.2; 63.5 | |
| SCN3B-4 | CTG CCC TGT CCC TAA CTG G | TTC CCT GTC CAC AGA GAG C | 358 | 2 | 1 | 57.2 to 67.2; 63.5 | |
| SCN3B-5 | TCC AAT GAC GGC TCT AGG T | GAG CAA GCA TTC TGA AGG TG | 258 | 2 | 1 | 55.3 to 65.3; 59.5 | |
| SCN3B-6 | CTC CTG TGC CCT GCT CCT T | ACA ACC TGC CAT CCA CAT TC | 219 | 2 | 1 | 53.7 to 63.7; 61.5 | |
| SCN4B-1 | GCT GTG CCC AGT ATC CCA T | CCA CCA TCC TCA TTC CGT G | 241 | 2* | 1 | 47.2 to 57.2; 64 | 54.7 to 64.7; 67 |
| SCN4B-2 | CCC GAG GTT GGC ACT GAG G | GGA CCA GAG CGT AGG AGG C | 373 | 2* | 1 | 58.5 to 68.5; 62 | |
| SCN4B-3 | TCT CGG CTA CTT TCT CAC CC | CCT CCC AAG TCC TTC CCA C | 320 | 2 | 2 | 57.3 to 67.3; 61 | |
| SCN4B-4 | GCT CCA GGT TGA CTC TTG CT | GCT GCT GGG AGG ACA GGA G | 326 | 2 | 2 | 57.4 to 67.4; 61.5 | |
| SCN4B-5 | TCC CCC TAC TCT TGC TCC T | GGA CTC TGG TTT CTT GTG CC | 294 | 2 | 2 | 56.5 to 66.5; 63 |
Polymerase chain reaction and other reactions were performed in 20-μl volumes with 50 ng of DNA, 16 pmol of each primer, 200 μM of each dNTP, 50 mmol/l KCl, 10 mmol/l Tris-HCl (pH 8.3), and 1.0 U of Amplitaq Gold (Applied Biosystems, Branchburg, NJ). PCR indicates polymerase chain reaction; DHLPC, denaturing high-performance liquid chromatography.
8% dimethyl sulfoxide was added to the reaction mixture. Polymerase chain reaction amplification was performed with a DNA Engine Tetrad thermal cycler.
Thermal cycling method 1: 94°C for 5 minutes, followed by 5 cycles of 94°C for 20 s, 64°C for 20 s, and 72°C for 30 s; additional 35 cycles of 94°C for 20 s, 62°C for 20 s, 72°C for 30 s, and a final extension of 72°C for 10 minutes. Thermal cycling method 2: 94°C for 15 minutes, followed by 35 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, and a final extension of 72°C for 10 minutes.
DHPLC was performed with a 5% buffer B/minute gradient. The start and stop % buffer B followed by the temperature at which the gradient was performed is indicated in the table.