A new approach to touch down method using betaine as co-solvent for increased specificity and intensity of GC rich gene amplification

• Published in: Gene 2012-04, Vol.497 (2), p.269-272
• Main Authors: Pratyush, Daliparthy D., Tiwari, Shalbha, Kumar, Ashok, Singh, Surya K.
• Format: Article
• Language: English
• Subjects: annealing; Betaine; Betaine - chemistry; Co-solvents; GC rich; GC Rich Sequence; Gene Amplification; genes; IRS2; melting point; Melting temperature (Tm); new methods; Nucleic Acid Amplification Techniques - methods; polymerase chain reaction; Polymerase Chain Reaction - methods; Sensitivity and Specificity; Solvents - chemistry; Temperature; Touchdown PCR
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/j.gene.2012.01.031

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. ► Initial annealing temperature below the primers Tm. ► Amplicon yield could effectively serve for RFLP, blotting and DNA sequencing. ► It is less time taking. ► Economic, requiring lower volumes of reaction mixture. ► Absence of spurious products that impede during RFLP and DNA sequencing.