Mj mini gradient pcr apparatus

Three DNA segments encompassing the studied SNPs were amplified using polymerase chain reaction (PCR). All primers are listed in S2 Table. PCR was performed using an MJ Mini gradient PCR apparatus (Bio-Rad, Hercules, CA, USA) in 20-μl reaction mixes containing 200 ng of genomic DNA, 0.2 mM dNTPs, Taq buffer with KCl (1:10 dilution), 1.5 mM MgCl₂, 0.2 mM forward and reverse primers, and 1 unit of Taq polymerase (Fermentas, Vilnius, Lithuania). The PCR conditions are shown in S3 Table. The resulting DNA fragments were purified with a gel extraction kit (Gel-Out kit; A&A Biotechnology, Gdynia, Poland) and sequenced using the amplification primers. The genotypes (P^1NORP², P¹P², P^1NORP¹, P¹P¹, and P²P²) were assigned based on the SNP that best correlated with the P₁/P₂ blood typing and the c.631C/G status. Nucleotide differences between hetero- and homozygotes for individual P₁/P₂-related SNPs are shown in S1 Table. The pp genotype of the sole p individual in the cohort was confirmed previously [21 (link)].

Genomic DNA was isolated from livers of sacrificed pigeons using Quick Blood DNA Purification Kit (EURx, Gdansk, Poland) according to the protocol provided by the supplier. Primers and conditions used in the PCR reaction for amplification of the A4GALT gene are listed in Supplementary Table S3A,B. PCR was performed using an MJ Mini gradient PCR apparatus (Bio-Rad, Hercules, CA, USA) in 100 µL reaction mixes containing 200 ng of pigeon genomic DNA (template), 0.2 mM dNTPs, HF Taq buffer with MgCl₂ (20× dilution), 0.2 mM forward and reverse primers, and 1 unit of HF Taq polymerase (Thermo Fisher Scientific, USA). The PCR products were purified using Gel-Out (A&A Biotechnology, Gdansk, Poland). The PCR products were further amplified using primers with restriction sites: XhoI (F) and NotI (R) (Thermo Fisher Scientific, Waltham, MA, USA) and cloned into the pCAG vector (kindly provided by Prof. Peter W. Andrews, University of Sheffield, Sheffield, UK) [18 (link)]. To test for the presence of A4GALT paralogs, the PCR products were digested with PaeI and HincII (Thermo Fisher Scientific, Waltham, MA, USA); 500 ng of purified PCR products were digested in 37°C for 6 h and analyzed by electrophoresis in 1% agarose gel with MIDORI green dye (Nippon Genetics Europe, Duren, Germany).