Supporting Information s33

Supporting information

“Multi-allele dipstick assay for visual genotyping of four novel SIRT1 gene variant alleles as candidate biomarkers for sporadic Parkinson disease”

Stella Koulouliaa, Marifili Lazaridoua, Theodore K. Christopoulosb,c, and Penelope C. Ioannoua* aLaboratory of Analytical Chemistry, Department of Chemistry, Athens University, Athens 15771, Greece. bDepartment of Chemistry, University of Patras, Patras 26500, Greece. cFoundation for Research and Technology Hellas, Institute of Chemical Engineering and High Temperature Chemical Processes (FORTH/ICE-HT), Patras 26504, Greece.

*Corresponding author. Phone: +30 210-7274574, Fax: +30 210-7274750. E-mail: [email protected]

1 Materials and methods

Construction of recombinant mutant DNA fragments Due to the lack of samples carrying the variant alleles, three recombinant DNA samples carrying one, three and four variant bases were constructed and used as controls. Recombinant DNA fragment carrying all the four SNPs (g.69644133G>A, g.69644213G>A, g.69644219G>A and g.69644351G>A) was constructed by sequential insertion of variant bases using mutagenic five-step PCR (PCR-A, PCR-B, PCR-C, PCR-D and PCR-E) (Fig. S1). PCR-A, PCR-B and PCR-C were carried out using the primer pairs F1(MUT)−R1(MUT2), F2’(MUT2)−R2(MUT), and F3(MUT)−R3(MUT), respectively, in a total volume of 50 μL, containing 1 U Robust Kapa 2G Fast DNA polymerase, 1x PCR buffer, 1.5 mM MgCl2, 200 μΜ dNTPs, 0.25 μΜ each of the appropriate primer and 50- 100 ng genomic DNA. The thermal cycling protocol consists of the following steps: 95 oC/ 3 min followed by 35 cycles of 95 oC/ 30 s, 62 oC/ 30 s (for fragments A, B) or 60 oC/ 30 s (for fragment C), 72oC/ 30 s, and a final extension step at 72oC for 2 min. Fragments A (219 bp), B (155 bp) and C (146 bp) were purified from a 1.2% agarose gel using the NucleoSpin extraction kit. An equimolar mixture of the purified B and C fragments (~200 fmol each fragment) were joined to give fragment D (284 bp) by means of a PCR-like reaction without primers. The thermal cycling protocol included the following steps: 95 oC/ 3 min followed by 40 cycles of 95 oC/ 30 s, 62 oC/ 30 s, 72oC/ 30 s and a final extension step at 72oC για 2 min. Subsequently, a 1 μL aliquot of fragment D was amplified by PCR using primer pair F2’(MUT2) and R3. Fragment D was purified from a 1.2% agarose gel using the NucleoSpin extraction kit. An equimolar mixture of the purified D and A fragments (~200 fmol each) were joined to give fragment E (481 bp) by means of a PCR-like reaction without primers. The thermal cycling protocol included the following steps: 95 oC/ 3 min followed by 40 cycles of 95 oC/ 30 s, 62 oC/ 30 s, 72oC/ 30 s and a final extension step at 72oC για 2 min. The 481 bp fragment containing all four polymorphisms was amplified by PCR using primer pair F0 and R0 and the thermal cycling protocol was 95 oC/ 3 min followed by 35 cycles of 95 oC/ 30 s, 65 oC/ 30 s, 72oC/

2 30 s and a final extension step at 72oC για 8 min. Then, fragment E was purified from a 1.2% agarose gel using the NucleoSpin extraction kit. The same procedure as above was followed for the construction of a variant DNA fragment containing three of the four polymorphisms (g.69644133C>G, g.69644213G>A and g.69644351G>A) using primer pair F1(MUT) and R1(MUT) to generate fragment A’ (219 bp) flanking the g.69644133C>G and g.69644213G>A polymorphisms and primer pair F2(MUT) and R2(MUT) to generate fragment B’ (155 bp) flanking the g.69644213G>A and g.69644351G>A polymorphisms. Fragments C, D’ and E’ were generated as described above. A third variant DNA fragment (481 bp) carrying only one of the four polymorphisms (g.69644133C>G) was also constructed using primer pair F1(MUT) and R3. PCR was carried out in a total volume of 50 μL, containing Robust Kapa

2G Fast DNA polymerase 1 U/reaction, 1x PCR buffer, 1.5 mM MgCl 2, 200 μΜ dNTPs, 0.25 μΜ of the appropriate primer and ~50 ng genomic DNA. The thermal cycling protocol included the following steps: 95 oC/ 3 min followed by 35 cycles of 95 oC/ 30 s, 60 oC/ 30 s, 72oC/ 30 s, and a final extension step at 72oC for 8 min. The fragment was purified from a 1.2% agarose gel using the NucleoSpin extraction kit. Normal and variant DNA fragments for SIRT1 polymorphisms were PCR amplified using primer pair F0 and R0 and were cloned into the vector pDrive (3.85 Kb) by means of the Qiagen PCR cloning kit from MBI Fermentas (Vilnius, Lithuania) according to the manufacturer’s instructions. The insertion of the polymorphisms into the synthetic sequences was confirmed by DNA sequencing (Fig. S2).

3 Supporting information, Fig. S1

Outline of the series of the amplification reactions involved in the construction of recombinant DNA fragments in the promoter region of SIRT1 gene carrying four, three and a single polymorphisms as described in the Experimental Section.

Supporting information, Fig. S2

DNA sequencing data for a wild-type sample (N) and recombinant DNA (M) carrying four variant bases. The arrows highlight the positions of the inserted variant bases. F0 and R0 are the primers used for DNA sequencing.

4 5 Supporting information, Fig. S3

Optimization studies. (a) Effect of the number of PEXT cycles. (b) Effect of Mg +2 concentration in PEXT reaction. (c) Effect of formanide concentration in running buffer. (d) Effect of the amount of PCR amplified product in PEXT reaction. For the position of each variant-specific probe see cartoon on the left side of the figure.