Disease Markers / 2019 / Article / Tab 2

Research Article

Glutathione Transferase P1 Polymorphism Might Be a Risk Determinant in Heart Failure

Table 2

PCR-RFLP: primer sequences, PCR conditions, restriction enzymes, and fragment lengths.

PolymorphismPrimer sequencesPCR protocolGel electrophoresis results

GSTA1C-69TF, 5-GCATCAGCTTGCCCTTCA -3
R, 5-AAACGCTGTCACCGTCCTG-3
Denature: 94°C for 3 min
Followed by 94°C for 30 s
Annealing: 56°C for 30 s
Extension: 72°C for 30 s
#cycles: 30
Final extension: 72°C for 10 min
Eam1104I incubation at 37°C overnight
GSTA1CC: 400 bp
GSTA1CT: 400 bp, 308 bp, and 92 bp
GSTA1TT: 308 bp and 92 bp

GSTP1Ile105ValF, 5-ACCCCAGGGCTCTATGGGAA-3
R, 5-TGAGGGCACAAGAAGCCCCT-3
Denature: 95°C for 10 min
Followed by 94°C for 30 s
Annealing: 59°C for 30 s
Extension: 72°C for 30 s
#cycles: 29
Final extension: 72°C for 10 min
Alw26I incubation at 37°C overnight
GSTP1Ile/Ile: 176 bp
GSTP1Ile/Val: 176 bp, 91 bp, and 85 bp
GSTP1Val/Val: 91 bp and 85 bp

GSTM1F, 5-GAACTCCCTGAAAAGCTAAAGC-3
R, 5-GTTGGGCTCAAATATACGGTGG-3
Multiplex PCR:
Denature: 94°C for 3 min
Followed by 94°C for 30 s
Annealing: 59°C for 30 s
Extension: 72°C for 45 s
#cycles: 30
Final extension: 72°C for 4 min
GSTM1active: 215 bp band
GSTM1null: absent band
GSTT1F, 5-TTCCTTACTGGTCCTCACATCTC-3
R, 5-TCACGGGATCATGGCCAGCA-3
GSTT1active: 480 bp band
GSTT1null: absent band
CYP1A1F, 5-GAACTGCCACTT CAGCTGTCT-3
R, 5-CAGCTGCATTTG GAAGTGCTC-3
Successful PCR reaction: 312 bp band
Unsuccessful PCR reaction: absent band

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