Question

If you have a GC reach template, what PCR additives you may use? What is their function? Can manipulating the thermal cycling condition help? How?

Answer 1

GC rich pcr amplification required an addictives of chemicals/reagents. We have to check at different temperature yo ensure whether it is worked or not. 2degree centegrades are used normally for different concentration.

Betaine(final concentration from 0.5-2.5m),DMSO(1-5%),ammonium saturated buffer(depends on concentration it will varies) are the most common reagents that are used for GC rich pcr amplification.

Betaine is used to reduce the duplex stability,DMSO and formamide react as a reducing the secondary structure.

TMAC ( tetra methyl ammonium chloride)is used for specificity of hybridization and it also increase the TM.

Yes.manipulation of thermal cycle should maintain certain limits of temperature for certain time.

In thermal cycling initial denaturation starts at 94°C for 30seconds.it is sufficient for all templates.it thermal cycle annealing,elongation-72°c,fill in extension-72°C,Hold-4°C are maintained along with suitable time (seconds).