1) 72 oC is low enough for a long primer to bind to single stranded template, so it should not be a problem for the template to turn back to double stranded form (as it have even higher Tm).
2) pfu, Taq etc. do not have strand displacement activity. if the templates are in double stranded form. there would no be any extension can happen.
so why?
the template is single stranded after the 94 denaturation step. Some does re anneal as the temperature drops but the primers are in huge molar excess compared with the template so kinetics favours a primer molecule finding a template strand before 2 template strands find each other. In practice after many ( 35 or more) cycles of a good pcr the template is abundant and does re anneal and can form concatomers or just anneal and the efficiency of the pcr will drop ( this latter after about 20cycles)
Taq type enzymes extend at 100-1000 bases a second so once the primer has annealed the extension of the double strand from the primer along the template is very quick. In those molecules where the template has re annealed with itself does not amplify. This is common with high GC templates which melt badly and re anneal at a very high temperature so these are more difficult to amplify
the template is single stranded after the 94 denaturation step. Some does re anneal as the temperature drops but the primers are in huge molar excess compared with the template so kinetics favours a primer molecule finding a template strand before 2 template strands find each other. In practice after many ( 35 or more) cycles of a good pcr the template is abundant and does re anneal and can form concatomers or just anneal and the efficiency of the pcr will drop ( this latter after about 20cycles)
Taq type enzymes extend at 100-1000 bases a second so once the primer has annealed the extension of the double strand from the primer along the template is very quick. In those molecules where the template has re annealed with itself does not amplify. This is common with high GC templates which melt badly and re anneal at a very high temperature so these are more difficult to amplify
Hi Paul Rutland, Thank you for your answer. It can explain all the obervations. And some reports said ssb can improve long PCR. so i guess it is true. are there mathematical models for the process? i am amplifying long and high GC DNA. how can keep the template in single stranded form during the extension step?
The main problems with high GC templates are re annealing at high temperature due to the high stability of triple hydrogen bonding and secondary and tertiary structure problems caused by complimentary C and G rich regions forming loops in the template with some double stranded dna at the neck of the loop. So in amplifying high GC dna you need to minimise these effects. Doing the pcr in final concentration oF 1Molar betaine minimises hydrogen bonding and makes the template melt easier and re anneal at a lower temperature allowing the primers to anneal and extend. Having high annealing temperature primers also helps a lot because they also do not allow the template to re anneal at low temperature. The Looping can be minimised with DMSO 1-8% in the reaction mix which keeps the molecule more linear. Addition of base analogues like 7 deaza dGTP and Inosine derivatives can replace G in the amplimer and help in maintaining an accessible structure in pcr methods. Betaine and dmso can be used together in the same reaction but too much dmso will kill the enzyme or make the primer annealing too specific and can inhibit pcr. For very long pcr (5 kb and longer) I Would trust commercial kits but I have always found dmso and betaine to work and have never needed to use ssb protein
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