Eco47III (AfeI) (10 U/µL)

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	To identify SNPs rs1726866 C/T and rs10246939 G/A, we subjected the 194 bp fragment 2Fmut/2Rmut at two different enzymatic digestions in the presence of endonuclease Eco47III and RsaI, respectively. A fragment modified in that manner shows the sequence AGCGCT for Eco47III and the sequence GTAC for RsaI. A 5 μL aliquot of the 194 bp PCR reaction fragment of was then mixed with a 15 μL solution containing 2 μL 10× buffer O (50 mM Tris-HCl,10 mM MgCl2, 100 mM NaCl, and 0.1 mg/mL BSA pH7.5), 2 μL Eco47III (10 U/mL) (ThermoFisher Scientific,Waltham, MA, USA), and 11 μL sterile deionized H2O. Similarly, a 5 μL aliquot of the same PCR reaction fragment was mixed with a 15 μL solution containing 2 μL 10× Tango buffer (33 mM Tris-acetate, 10 mM Mgacetate, 66 mM K-acetate, and 0.1 mg/mL BSA; pH 7.9), 2 μL RsaI (10 U/mL) (ThermoFisher Scientific), and 11 μL sterile deionized H2O. Each reaction (Eco47III and RsaI) reaction mixture was mixed and then incubated at 37°C for 2 h (Figure 3). The digest (10 μL) was mixed with 3 μL of loading buffer and electrophoresed on a 10% vertical polyacrylamide gel. Silver nitrate and ethidium bromide staining were carried out according to the methods of Herring et al. (1982) and Sambrook et al. (1989), respectively.

Protocol tips

To identify SNPs rs1726866 C/T and rs10246939 G/A, we subjected the 194 bp fragment 2Fmut/2Rmut at two different enzymatic digestions in the presence of endonuclease Eco47III and RsaI, respectively. A fragment modified in that manner shows the sequence AGCGCT for Eco47III and the sequence GTAC for RsaI. A 5 μL aliquot of the 194 bp PCR reaction fragment of was then mixed with a 15 μL solution containing 2 μL 10× buffer O (50 mM Tris-HCl,10 mM MgCl2, 100 mM NaCl, and 0.1 mg/mL BSA pH7.5), 2 μL Eco47III (10 U/mL) (ThermoFisher Scientific,Waltham, MA, USA), and 11 μL sterile deionized H2O. Similarly, a 5 μL aliquot of the same PCR reaction fragment was mixed with a 15 μL solution containing 2 μL 10× Tango buffer (33 mM Tris-acetate, 10 mM Mgacetate, 66 mM K-acetate, and 0.1 mg/mL BSA; pH 7.9), 2 μL RsaI (10 U/mL) (ThermoFisher Scientific), and 11 μL sterile deionized H2O. Each reaction (Eco47III and RsaI) reaction mixture was mixed and then incubated at 37°C for 2 h (Figure 3). The digest (10 μL) was mixed with 3 μL of loading buffer and electrophoresed on a 10% vertical polyacrylamide gel. Silver nitrate and ethidium bromide staining were carried out according to the methods of Herring et al. (1982) and Sambrook et al. (1989), respectively.

Publication protocol

"A 5 μL aliquot of the PCR reaction fragment of
203 bp containing the rs713598 G/C SNP was mixed
with a 15 μL solution containing 2 μL 10× NE buffer
(50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM
dithiothreitol, pH 7.9), 0.2 μL HaeIII (10,000 U/mL)
(Sigma-Aldrich, St. Louis, MO, USA), and 11 μL
sterile deionized H2O, then incubated at 37°C for
2 h (Figure 3). To identify SNPs rs1726866 C/T and
rs10246939 G/A, we subjected the 194 bp fragment
2Fmut/2Rmut at two different enzymatic digestions in
the presence endonuclease Eco47III and RsaI, respectively. A fragment modified in that manner shows the
sequence AGCGCT for Eco47III and the sequence
GTAC for RsaI. A 5 μL aliquot of the 194 bp PCR reaction fragment of was then mixed with a 15 μL solution containing 2 μL 10× buffer O (50 mM Tris-HCl,
10 mM MgCl2, 100 mM NaCl, and 0.1 mg/mL BSA pH
7.5), 2 μL Eco47III (10 U/mL) (ThermoFisher Scientific,
Waltham, MA, USA), and 11 μL sterile deionized H2O.
Similarly, a 5 μL aliquot of the same PCR reaction fragment was mixed with a 15 μL solution containing 2 μL
10× Tango buffer (33 mM Tris-acetate, 10 mM Mgacetate, 66 mM K-acetate, and 0.1 mg/mL BSA; pH 7.9),
2 μL RsaI (10 U/mL) (ThermoFisher Scientific), and
11 μL sterile deionized H2O. Each reaction (Eco47III
and RsaI) reaction mixture was mixed and then incubated at 37°C for 2 h (Figure 3). The digest (10 μL) was
mixed with 3 μL of loading buffer and electrophoresed
on a 10% vertical polyacrylamide gel. Silver nitrate and
ethidium bromide staining were carried out according
to the methods of Herring et al. (1982) and Sambrook
et al. (1989), respectively. The digested pUC18 DNA
HaeIII plasmid (Sigma-Aldrich) and Edu-PCR MW
Ruller (Bio-Rad Laboratories, Inc. Hercules, California,
USA) were used as an MW marker. All PCRs and digestions were conducted in triplicate for each DNA sample.
Figure 3 shows an example of the electrophoretic profiles obtained in the presence of the three SNPs of the
TasR38 gene. In order to verify the accuracy of the PCRrestriction fragment length polymorphism method, all PCR samples were sequenced by the Sanger method
with an ABI Prism 310 automated sequencer and for
more accuracy and better confirmation of results, the
genotyping was based on both the forward and reverse
strands. Translations of nucleotide sequences were performed using ExPASy translate routine software (http://
ca.expasy.org/). Allelic frequencies were determined
and statistical analysis was performed using the Chi
square (χ2
) test. Fisher’s method (Genepop software
version 4.0; http://kimura.univ-montp2fr/~rousset/
Genepop.htm) was used to test TAS2R38 genotype distribution and haplotype frequencies, as well as genotype
distribution and allele frequencies of the CA6 gene polymorphism rs2274333 (A/G) in terms of PROP status.
Genetic differences among the three taster groups based
on the distribution of the TAS2R38 and CA6 genotype
combinations were tested by the Markov Chain method
(Arlequin software version 3.1; http://cmpg.unibe.ch/
software/arlequin3)"

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