A new approach to touch down method using betaine as co-solvent for increased specificity and intensity of GC rich gene amplification.
Tissue specific genes that contain high GC segments are difficult to amplify by standard PCR. We report an improved method for successful amplification of gene segment that has >70% GC base pairs. This new method of touch down PCR differed by having an initial annealing temperature (Ta) 1.5°C below the primers melting temperature that descended 0.2°C per cycle for 20 cycles and continued thereafter at fixed Ta for next 15 cycles. Different co-solvents were tested with this method to improve the result and betaine proved better than the other co-solvents. This new method is economical, fast and specific in amplifying GC rich region of other genes also.
Department of Endocrinology and Metabolism, Banaras Hindu University, Uttar Pradesh, India.
Daliparthy D
DD
Pratyush
Shalbha
S
Tiwari
Ashok
A
Kumar
Surya K
SK
Singh
D001622:Betaine / Q000737:chemistry*; D020862:GC Rich Sequence; D005784:Gene Amplification; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012997:Solvents / Q000737:chemistry; D013696:Temperature
D012997:Solvents; D001622:Betaine