Genotype for 15 non-synonymous single-nucleotide polymorphisms (SNPs) in the P2X7 gene v1
Authors: KCS Stefani Kelly , Dresh Martinhago Ciro , Diniz Fernandes Tulio 
Published: February 2020

Biochemical analysis DNA was obtained by buccal swabs of the patients; each cheek was swabbed ten times using a cytology brush. Two brushes were used per patient, one for each cheek. Each brush was transferred to a properly labelled Eppendorf tube containing approximately 1 mL of 0.9% saline; the samples were stored at room temperature. Mouthwash was used because it is the least invasive and most practical method of obtaining DNA. The brush was stirred vigorously in the solution. The brush was then removed using tweezers, and the samples were processed. DNA was extracted automatically by column extraction using the QIACube method. This stage involved DNA extraction from the sample, nucleic acid stabilisation, removal of amplification inhibitors, and concentration of the sample in a volume of aqueous solution compatible with the subsequent steps in the protocol. Proteinase K and lysis buffer were added to 200 µL of the total sample. The lysis buffer contains high concentrations of salts, including guanidine thiocyanate and guanidine isothiocyanate, that promote cell lysis, the degradation of proteins and membrane debris, and DNA adsorption to the silica membrane. The membrane was then washed under pH and salt conditions appropriate for eluting DNA in high yield and purity. The yield from a 200-µL sample was 3 to 12 µg of DNA with an A260/A280 ratio of 1.7–1.9. Genotyping Genetic polymorphisms were analysed by a group of researchers who performed the sample processing and were blind to the groups to which the patients belonged. The study population was genotyped for the 13 exons containing 15 non-synonymous SNPs in the P2XR7 receptor gene (chromosome 12q24). These SNPs were selected based on their functional effects on this gene and their availability in the database of non-synonymous SNPs. Genotyping was performed using polymerase chain reaction (PCR). Subsequently, quantitative genotyping, SNP detection, and allelic discrimination were performed.SNP genotyping was performed using an extension reaction with two types of primer design: single-base extension and allele-specific oligonucleotides. The primers used in PCR were designed using Batch Primer3 software.The UCSC In-Silicone PCR®tool was used to confirm the sequence of each primer pair specific to the genomic region to be amplified.The PCR primers were sequenced using the Sanger and Long Range methods; all oligonucleotides were synthesised by Thermo Fisher Scientific® . The primer sequence used in SNP1 sequencing was designed using the Sanger method and covered the intronic region between exons 1 and 2. PCR was performed using the AmpliTag Gold® Fast PCR Master Mix (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol in a total volume of 10 µL. A thermocycler (Applied Biosystems) was used for amplification. The amplification product was purified using the ExoSAP-IT purification kit (USB Corporation, Cleveland, OH, USA) to remove unincorporated primers and free nucleotides according to the manufacturer’s protocol. The purified products were sequenced in an AB3500 sequencer (Applied Biosystems) using the BigDyeTerminator v3.1 sequencing kit (Applied Biosystems). The results were analysed using the software Seqscape version 2.7 (Applied Biosystems). The primer pair used in SNP2-15 sequencing was synthesised using two long-range PCR reactions. The Platinum Taq DNA Polymerase High Fidelity kit at a final concentration of 1´(Invitrogen) was used under the following conditions: 25 µL of 1´PCR buffer, 0.2 mM of each dNTP, 0.2 µM of each primer, 2 mM MgSO4, 2.5 µL of DMSO (Merck), 0.5 U of Platinum Taq DNA Polymerase High Fidelity, and 100 ng of DNA. The programme used for long-range PCR 1 was 94 °C for 2 min, 10 cycles of 94 °C for 10 s, 63 °C for 15 s, and 68 °C for 14 min, 25 cycles of 94 °C for 10 s, 60 °C for 15 s, and 68 °C for 14 min (with an increase of 20 s/cycle), and final extension at 68 °C for 7 min. The programme used for long-range PCR 2 was 94 °C for 2 min; 10 cycles of 94 °C for 10 s, 65 °C for 15 s, and 68 °C for 14 min; 25 cycles of 94 °C for 10 s, 63 °C for 15 s, and 68 °C for 14 min (with an increase of 20 s/cycle); and final extension at 68 °C for 7 min. The PCR products were analysed on 2% agarose gels, and the amplification yield was calculated. The minisequencing reactions employed for SNP genotyping used the PCR products of the SNP-flanking regions co-amplified in multiple reactions (multiplex) as a template. Before initiating the minisequencing reaction, the PCR products were cleaned to remove excess primers and unincorporated dNTPs using the ExoSAP-IT purification kit (Amersham Biosciences, Uppsala, Sweden). For this purpose, 3 µL of the PCR product was incubated with 1.5 µL of ExoSAP-IT reagent for 15 min at 37 °C and for 15 min at 80 °C for enzymatic inactivation. The SNPs were then genotyped using polychromatic minisequencing (SNaPshotTM Multiplex System, Applied Biosystems) . Multiplex minisequencing was performed using a reaction volume of 10 µL consisting of 2.5 µL of the purified PCR product, 5 µL of SNaPshot™ kit Reaction Mix, 1.5 µL of a primer mix (5 pM each primer), and Milli-Q water q.s. The reaction was conducted according to the following protocol: 25 denaturation cycles at 96 °C for 10 s, annealing at 50 °C for 5 s, and extension at 60 °C for 30 s. The purified products were sequenced on an ABI PRISM 3500 Genetic Analyzer (Applied Biosystems) sequencer using 1.5 µL of the final amplicon, 10 µL of HiDi™ formamide (Applied Biosystems), and 0.25 µL of GeneScan-120 LIZ (Applied Biosystems). The electrophoresis parameters were as follows: capillary length 36 cm, injection voltage 1.2 kV, injection time 18 s, voltage 15 kV, and running time 800 s. The results were analysed using the software GeneMapper version 5 (Applied Biosystems). Sequencing quality was assessed by conventional sequencing to confirm the obtained results.