Genotype for 15 non-synonymous single-nucleotide polymorphisms (SNPs) in the P2X7 gene v1 (protocols.io.bcf2itqe) [dataset]

Kelly Stefani, Ciro Dresh, Tulio Diniz
2020 protocols.io  
1 1 1 1 Bi oche mi ca l a na l ysi s Bi oche mi ca l a na l ysi s 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
more » ... 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. G e notypi ng G e notypi ng 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 Bi oche mi ca l a na l ysi s Bi oche mi ca l a na l ysi s 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 [30] . 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 [31] . 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.
doi:10.17504/protocols.io.bcf2itqe fatcat:pizcsdvdvzcgrbv2jsbtmdy5du