Denaturing High-Performance Liquid Chromatography Analysis

APPENDIX

PCR conditions

Thirty-five cycles of 30s at 950C, 1 min at 670C and 2 min at 720C was performed using a PTC-225

Peltier Thermal Cycler (MJ Research, Watertown, MA) with BioTaq (Bioline, London). 8 primers were used for direct sequencing (Table 1).

Denaturing high-performance liquid chromatography analysis

Unpurified PCR products at 3:1 ratio with a wild-type reference before subjected to a 3 min, 95 °C denaturing step followed by gradual reannealing from 95–65 °C over 30 min. l of each mixture was loaded onto a DNASep column (Transgenomic, Omaha, Neb., USA). and the amplicons were eluted in 0.1 M triethylammonium acetate, pH 7, with a linear acetonitrile gradient at a flow rate of 0.9 ml/min2. Heteroduplex mismatches were recognized by the appearance of aberrant patterns in the elution profiles under appropriate temperature conditions, which were calculated by the WAVEMaker software of the Wave Nucleic Acid Fragment Analysis system HSM device.

SHP G171A genotyping

A primer (5’-CTC ACC GGG GTT GAA GAG GAT GGg C-3’) was designed to mutagenise a nucleotide 2 base pairs downstream from where the variation occurs, forcing a restriction enzyme site.

Thus the wild-type (G171) allele could be digested with Hae III and this became the basis of a rapid screening assay. The other primer sequence was derived from the complementary strand (5’-CGG

TGC AGT GGC TTC AAT GCT GTC-3’). PCR was carried out under standard conditions. Four cycles of 30s at 950C, 30s at 720C and 30s at 720C with the annealing temperature decreasing 10C per cycle followed by thirty-three cycles of 30s at 950C, 30s at 680C and 30s at 720C were performed to amplify the DNA fragment for the digestion. After digestion with Hae III (New England Biolabs,

Beverly, MA), the digested PCR product yielded 2 fragments, 79 and 25 bp in size. The undigested fragment was 104 bp in length. These fragments were resolved by electrophoresis through 3% (wt/vol) agarose gels (GibcoBRL, Paisley, U.K.), detected with ethidium bromide staining and visualised under ultraviolet illumination.

SHP -195CTGAdel genotyping

The -195CTGAdel variants could be distinguished by inspecting the amplified product on the agarose gel. The products amplified from the heterozygous individuals using the pair of primers for the amplicon 3 were resolved as double-band on a 3% agarose gel, whereas the amplified products of the homozygous individuals were detected as a single band. In order to distinguish the individuals who are homozygous for the -195CTGAdel allele from those homozygous for the wide type allele, two separated rounds of PCR were carried out . In the first round of PCR, some DNA known to be homozygous for the wild type allele was added into the reaction mix so that the amplified products of the individuals homozygous for the -195CTGAdel allele could be visualised as double-band on the gel. These individuals were scored as the -195CTGAdel allele carriers. The variant carriers were then subject to the second round of PCR, in which no other source of DNA was introduced. The genotype of the individuals whose amplified product were resolved as a single band on the gel in the second round was scored as homozygous for the -195CTGAdel allele. TABLE 1 Primer pairs used to amplify fragments for DHPLC.

Amplicon Primer Pair (5’-3’) Size 1 F: GAG GAG GAG GCC TCC TAG ATT G 270 R: CAG GTG CAC AAT CAG CAG GTG

2 F: CCT GGC TTA GCA AAA GCC CT 214 R: GAT ATC ACC TCA GTC AAT GAA GT

3 F: CAC CAA TGG GGA CAC CTG CTG A 273 R: ACT CCA GAA GTC ATG TTC ATT G

4 F: CCA CCA CTT CCC CAC CAT TCC T 268 R: GAG GCT GGA GCT CAG AAG TGC GT

5 F: TAG CAC CCA CAG CGC AGA ACA 263 R: CAC TGT CTT GGC CAG AAC ATC C

6 F: CTC CAG CCT CAA GGC TGT CC 251 R: ACC TCA AAG GTC ACA GCA TCT

7 F: GCC TTC CTC AGG AAC CTG CCA T 328 R: GAG GAC CCA ATG AGA TAA CAG ATA TCA 8 F: CAC CAG CCC TCT TCT CCC TCT 336 R: GCC AGG CTG AAT CAG CAC TGC CA