The required part of genomic DNA was amplified using primers listed in S1 Table to determine the presence or the absence of the mutant allele for each analysed gene.
PCR mixture for each amplification reaction contained 2–2.5 mM MgCl2, 1x Dream Taq buffer (Thermo Fisher Scientific), 1 μM1 of both forward and reverse primer, 0.25 mM dNTPs (Thermo Fisher Scientific), 1 U Dream Taq polymerase (Thermo Fisher Scientific), approximately 50 ng of template DNA and H2O.
We used PCR followed by the restriction fragment length polymorphism (RFLP) analysis to detect alleles of CMR1, DM, PRCD and SHT alleles. The restriction enzyme digestion was performed in a 20 μl reaction mixture which consisted of 2 U of the restriction endonuclease (HphI—CMR1, Eco57I - DM, SfaNI—HUU, AlwI and RsaI—PRCD, FD-Eco91I - SHT) (Thermo Fisher Scientific), 1x supplied buffer, 10 μl PCR product and distilled water. Fragments were separated by size using electrophoresis on 1.5% agarose gel or 10% polyacrylamide gel, depending on the product length.
Sequencing was used for HSF4 mutation screening and for verifying the results of RFLP methods. The five samples were chosen randomly from each genotype available, and the sequencing data were compared with the results of the RFLP analysis.
The PCR products were directly sequenced after ExoSAP-IT (Applied Biosystems) treatment using PCR primers with the ABI BigDye Terminator Sequencing Kit 3.1 (Applied Biosystems) on an ABI 3500 capillary sequencer. Sequence data were analysed with Vector NTI Advance 7.0 (Invitrogen).
PCR mixture for each amplification reaction contained 2–2.5 mM MgCl2, 1x Dream Taq buffer (Thermo Fisher Scientific), 1 μM1 of both forward and reverse primer, 0.25 mM dNTPs (Thermo Fisher Scientific), 1 U Dream Taq polymerase (Thermo Fisher Scientific), approximately 50 ng of template DNA and H2O.
We used PCR followed by the restriction fragment length polymorphism (RFLP) analysis to detect alleles of CMR1, DM, PRCD and SHT alleles. The restriction enzyme digestion was performed in a 20 μl reaction mixture which consisted of 2 U of the restriction endonuclease (HphI—CMR1, Eco57I - DM, SfaNI—HUU, AlwI and RsaI—PRCD, FD-Eco91I - SHT) (Thermo Fisher Scientific), 1x supplied buffer, 10 μl PCR product and distilled water. Fragments were separated by size using electrophoresis on 1.5% agarose gel or 10% polyacrylamide gel, depending on the product length.
Sequencing was used for HSF4 mutation screening and for verifying the results of RFLP methods. The five samples were chosen randomly from each genotype available, and the sequencing data were compared with the results of the RFLP analysis.
The PCR products were directly sequenced after ExoSAP-IT (Applied Biosystems) treatment using PCR primers with the ABI BigDye Terminator Sequencing Kit 3.1 (Applied Biosystems) on an ABI 3500 capillary sequencer. Sequence data were analysed with Vector NTI Advance 7.0 (Invitrogen).
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